Cholestosome vesicles for incorporation of molecules into chylomicrons

ABSTRACT

The present invention is directed to a cargo-loaded cholesteryl ester nanoparticle with a hollow compartment (“cholestosome”) consisting essentially of at least one non-ionic cholesteryl ester and one or more encapsulated active molecules which cannot appreciably pass through an enterocyte membrane in the absence of said molecule being loaded into said cholestosome, the cholestosome having a neutral surface and having the ability to pass into enterocytes in the manner of orally absorbed nutrient lipids using cell pathways to reach the golgi apparatus. Pursuant to the present invention, the novel cargo loaded cholestosomes according to the present invention are capable of depositing active molecules within cells of a patient or subject and effecting therapy or diagnosis of the patient or subject.

RELATED APPLICATIONS

This application claims the benefit of priority of provisionalapplication no. U.S. 61/783,003, filed Mar. 14, 2013, of identicaltitle, the entire contents of which are incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention is directed to a cargo-loaded cholesteryl ester(lipid) nanoparticle with a hollow compartment (“cholestosome”)consisting essentially of at least one non-ionic cholesteryl ester andone or more encapsulated active molecules which, in the absence ofencapsulation in cholestosomes cannot appreciably pass through anenterocyte membrane in the absence of said molecule being loaded intosaid cholestosome, the cholestosome having a neutral surface and havingthe ability to pass into enterocytes in the manner of orally absorbednutrient lipids using cell pathways to reach the golgi apparatus.Pursuant to the present invention, the novel cargo loaded cholestosomesaccording to the present invention are capable of depositing activemolecules within cells of a patient or subject and effecting therapy ordiagnosis of the patient or subject. The compositions according to thepresent invention are substantially more active than compositions of theprior art which do not use cholesteryl ester nanoparticles.

In one embodiment, the invention provides a cholesteryl esternanoparticle pharmaceutical composition comprising an active molecule(“cargo-loaded cholestosome”), for example, a pharmaceutically-activeagent (which term includes therapeutic and diagnostic agents) which isencapsulated by a surface layer of neutral charge comprising one or morecholesteryl esters produced from cholesterol and one or more saturatedor unsaturated fatty acids. The cholestosomes according to the presentinvention encapsulate one or more different active molecules of widevariety of size and weight, especially pharmaceutical active moleculeswhich are difficult to deliver using prior art methods, includingliposomes.

Pursuant to the present invention, the cargo-loaded cholestosomes, afteradministration to a patient or subject, are incorporated intact intochylomicrons (generally, after uptake into intestinal enterocytes) toproduce a nanoparticle containing chylomicrons and said nanoparticlecontaining chylomicrons are delivered into the lymphatics andsubsequently into arterial blood and to all cells receiving saidarterial blood supply, whereupon after docking of the chylomicrons withcells, the cholestosome is delivered intact into said cells, whereinsaid cholestosome is disassembled, releasing the encapsulated activemolecules inside the membrane of said cells. The impact of the presentinvention is to directly deliver active molecules inside cells to effecttherapy or diagnosis.

Cholesteryl esters are selected for the composition of the nanoparticle,based on their reactivity with cholesterol transporters on the surfaceof intestinal (duodenal) enterocytes, which facilitates their rapid andcomplete uptake into the enterocytes. Once inside, cholesteryl esternanoparticles offer the added benefit of protection of the contents ofthe nanoparticle during chylomicron formation inside the enterocyte.Additional favorable properties of the cholesteryl ester components ofthe nanoparticle are 1) their surface neutral charge allowing theenterocytes to see these particles as food components, 2) their entirecomposition of the cholestosome from safe dietary ingredients, and 3) inparticular on their potential to “pack” with each other and therequirements of the pharmaceuticals to be incorporated in thenanoparticles themselves. Liposome manufacturing technology teaches awayfrom the use of cholesteryl esters in vesicles with neutral surfaces. Infact, if nanoparticles are made of phospholipids in the manner ofliposomes disclosed in the art, every one of the beneficial features ofthe present invention is lost.

Pharmaceutical compositions and oral methods of treatment of theinvention, when encapsulated with said cholesteryl esters, enablechylomicron-targeted intracellular delivery of a variety of activeingredients that are, in an unprotected state, ineffective due todegradation in vivo. For example, the invention enables effectivedelivery of macromolecules useful in the treatment ofinflammation-associated metabolic disorders as defined herein, vaccinesto specific sites in the body, genetic materials inside cells where theyact in the ribosomes and nuclei, and even topical delivery on the skinwith the potential for passage of the skin barrier in some specificembodiments. Other methods of treating disease states and/or conditionsusing compositions according to the present invention are alsodisclosed. Virtually any active molecule can be delivered efficientlyinto target cells of a patient or subject resulting in effective therapyunmatched by delivery methods of the prior art. Methods of treatingdisease states and conditions by administering compositions according tothe present invention to a patient in need represent additionalembodiments according to the present invention. Effective dosages ofcompositions for methods of treatment embodiments according to thepresent invention may range from as little as one mg or less up to onegram or more per day. Other effective dosages will depend on the sizeand age of the patient or subject, the general health of the patientamong a number of other facts. Dosages contemplated within the range ofless than about 0.001 mg/kg/day up to about 100 mg/kg/day or more withranges of about 0.01 mg/kg/day to about 25 mg/kg/day being more oftenutilized.

BACKGROUND OF THE INVENTION

There are many new therapeutic products where a large protein or othermacromolecule is serving a role as a therapeutic or diagnosticsubstance. For treatment of chronic conditions, there is a high interestin delivery of large molecules via non intravenous routes such assubcutaneous injection, in order to improve patient convenience andcompliance. Oral administration of peptides (including polypeptides suchas monoclonal antibodies), proteins, and DNA would be much moreconvenient and no less safe. However, many believe it is not possible toachieve oral absorption of large protein molecules in humans. Becauseorally administered molecules such as proteins, peptides and geneticmaterial are either digested in the gastrointestinal (GI) tract or failto diffuse across the cellular membrane of the enterocytes, or both, itis widely believed that parenteral delivery is the only reliable way toadminister such active ingredients. When given by the oral route,proteins are not absorbed intact by intestinal cells. Rather, they arebroken down by enzymes into amino acid constituents and thus most of thetherapeutic proteins produced by the biotechnology industry arecompletely susceptible to gastrointestinal degradation pathways.

The usual administration route, parenteral administration, is on theother hand suboptimal for macromolecular delivery for many reasons.Compared to oral administration, parenteral delivery is more expensiveand requires hardware and more highly trained personnel.

Even after parenteral administration, the macromolecules encounterproblems with passage of membranes. They are excluded from many targetcells, and as a result they circulate in blood until cleared or degradedbut may never successfully enter body cells. Macromolecules may fail topass regional barriers such as the blood brain barrier, effectivelypreventing targeting of macromolecules to selected organs and tissuessuch as brain. This may be an underlying reason for clinical trialfailure of many of the monoclonal antibodies against targets in theamyloid pathway to clear amyloid from the brain and their lack ofsufficient activity to reverse Alzheimer's disease. In general, thelarge size and lack of lipid solubility of these proteins may limit theintracellular effectiveness of an otherwise novel target monoclonalantibody.

Clearly, success with oral proteins depends on creation of novelformulations that overcome acid and/or enzymatic degradation in the GItract and then overcome low permeability across an intestinal enterocytemembrane, and finally overcome the current inability to pass into thecells on the other side.

Recent formulations that overcome only the gastrointestinal degradationproblems might achieve ˜5% absorption. This step is clearly importantbut insufficient, so it remains necessary to further improve the poorbioavailability of proteins with a novel means of taking up proteinsinto enterocytes, and this is disclosed herein for the first time.

Furthermore, the delivery means of the present invention is the first tosolve the next problem, that of intracellular delivery, by means of atransformative step performed on the nanoparticle, the incorporation ofthe lipid nanoparticle into chylomicrons with its molecular payloadintact. Successful incorporation into chylomicrons is only possible withthe use of herein disclosed cholesteryl esters to build the lipidnanoparticle.

Prior attempts to deliver macromolecules for oral absorption by theenterocytes have relied on encapsulation in nano sized particles. Mostof the work has been conducted with liposomes of varying composition.

As explained in the following excerpt from United States PatentApplication Document No. 20110229529, liposomes have not solved theaforementioned problems. “Liposomes have been widely used as a deliveryvehicle for small molecules; however, it remains difficult to achievehigh levels of encapsulation for many macromolecular drugs withinliposomes. Furthermore, many drug formulations leak from liposomes tooquickly to maintain useful drug delivery kinetics. While drug deliveryby micro- and nanoparticles can encapsulate proteins and small-moleculedrugs, this still typically yields very low total mass encapsulated drugper mass of particles, typically on the order of about 1:1000 to1:10,000 mass ratio, of in this case protein:phospholipid mixture (seefor example U.S. Pat. No. 7,662,405). In addition, the organic solventsused in polymer particle synthesis and hydrophobic/acidic environmentwithin these particles can lead to destruction of therapeutics. (See Zhuet al. Nat. Biotechnol. 2000 18:52-57.)”

There are other problems with use of liposomes even beyond theaforementioned small amount of encapsulation of water soluble proteinsor small molecules. Specifically, the contents of most liposomes arephospholipids, typically phosphatidylcholine. These nano sized lipidparticles are highly positively charged and thereby repelled by theouter membranes of enterocytes and also by cell membranes of peripheralcells.

Phospholipid based liposomes are thus not orally absorbed and are alsonot able to pass their contents into cells when injected parenterally.Thus no liposome of current composition is suitable for encapsulation ofproteins or peptides (including polypeptides such as monoclonalantibodies), and even it one could load enough molecule into theseparticles, they would not solve the oral absorption problem.Furthermore, no phosphatidyl choline based liposome can be incorporatedinto a chylomicron with its molecular payload intact.

Tseng and colleagues described these problems in 2007 (Tseng et al, J ofMedical and biological engineering 2007; 27: 29-34; the Tseng articlewas titled Liposomes incorporated with cholesterol for drug releasetriggered by magnetic field) and therein tested the hypothesis thatadding cholesterol to Phosphatidyl choline liposomes would alter theseproperties and improve loading. They found only modest improvement inloading, and there was not sufficient cholesterol to change the positivecharge of the outer surface. Of greater significance to them was theirobservation that increased cholesterol in the liposome prevented exit ofthe loaded molecules. “An increase of the cholesterol content inliposomes results in a dramatic decrease in membrane permeability fornon-electrolyte and electrolyte solutes. An optimized drug delivery vialiposomes requires the liposome carrier to ultimately become permeableand release the encapsulated drug on the targeted area, but it alsorequires high stability in the bloodstream” Thus entire the liposomalfield largely abandoned cholesterol as a component of liposomes, citinga deterioration in the molecular RELEASE properties of cholesterolcontaining liposomes and teaching the entire field away from theparticular nanoparticles of the present invention.

It should be noted in the present invention, that inventors have chosenthe high loading and slow release properties cholesteryl esters for thespecific purposes of protecting the molecule during its journey acrossmembranes of the GI tract enterocytes, then into chylomicrons, thenthrough the cell membranes. Unpacking of cholestosome encapsulatedproteins only occurs inside the body cells, which confers a greatadvantage to the disclosed delivery method over any current system. Wedisclose the analogy to the Trojan Horse, invented of course beforethere were patents, but not used heretofore for a drug delivery system.

It should also be noted that the disclosed process works as intendedonly with cholesteryl esters, as only these molecules are handled intactamong lipids all the way to intracellular delivery by chylomicrons.

Given the limitations of existing macromolecule therapies, the needcontinues to exist for formulations and treatments that administerpharmaceutically active macromolecules in a more convenient way such asorally, and the need continues for formulations that allow proteins andother molecules to enter cells. The use of one formulation to accomplishboth aspects is the primary subject of the present invention.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a cholesteryl esternanoparticle pharmaceutical composition comprising apharmaceutically-active agent (cargo-loaded cholestosome) which isencapsulated by a surface layer comprising one or more non-ioniccholesteryl esters. The cholesteryl esters used in the present inventionare produced from cholesterol (as defined herein) and one or moresaturated or unsaturated fatty acids as otherwise described herein,preferably a C₄-C₃₆ fatty acid, often a C₈-C₂₆ fatty acid, more often afatty acid selected from the group consisting Myristoleic acid,Palmitoleic acid, Sapienic acid, Oleic acid, Elaidic acid, Vaccenicacid, Linoleic acid, Linoelaidic acid, α-Linolenic acid, Arachidonicacid, Eicosapentaenoic acid, Erucic acid, Docosahexaenoic acid, Caprylicacid, Capric acid, Lauric acid, Myristic acid, Palmitic acid, Stearicacid, Arachidic acid, Behenic acid, Lignoceric acid, Cerotic acid or amixture thereof. The cholestosomes according to the present inventionare avidly taken up by the enterocytes of the gastrointestinal tract,and are rapidly transferred, along with their contents, intochylomicrons, thereby providing a means of transporting the encapsulatedmolecules directly into body cells, as well as incidentally andimportantly, bypassing hepatic first pass uptake pathways in theprocess. Quite unexpectedly, the presently claimed cargo-loadedcholesteryl ester vesicles are able to deliver a wide variety ofmolecules, including peptides (including polypeptides such as monoclonalantibodies) and proteins and other macromolecules, includingpolynucleotides such as DNA and RNA, macromolecular antimicrobial agents(anti-bacterial, anti-viral, anti-fungal, anti-parasitic and anti-prion)which vary greatly in size and molecular weight, into cells such thattherapy or diagnosis is effected.

In the present invention, the mass ratio of the active molecule (whichpreferably includes a pharmaceutically-active agent), to one or morecholesteryl esters is between about 4:96 to about 96:4, about 10:90 toabout 96:4, often about 10:90 to about 96:4, often about 20:80 to about90:10, about 20:80 to about 50:50, about 50:50 to about 96:4, about90:10 to about 96:4.

Physical Properties

In certain embodiments, the pharmaceutical composition is a unilamellarvesicle in which between about 10% to about 98%, about 20% to about 96%,often about 50% to about 96%, often about 90% to about 96% of thevesicle's volume is occupied by the pharmaceutically-active agent.

In another embodiment, an interdigitated alternating alkyl chain modelis used to maximize the mass ratio of the active molecule, including apharmaceutically-active agent to one or more cholesteryl esters byselecting the one or more cholesteryl esters based onpharmaceutically-active agent-cholesteryl ester functional groupinteraction. Example 2, infra describes formulation criteria whichensure optimal pharmaceutically-active agent-cholesteryl esterfunctional group interaction.

In another embodiment, the pharmaceutical composition is a cholestosomevesicle made by a process comprising reacting one or more of thecholesteryl esters in diethyl ether, removing the resultant organicphase under vacuum and introducing an aqueous phase.

In still another embodiment, cholesteryl esters are selected based ontheir reactivity with cholesterol transporters on the surface ofduodenal enterocytes and ability to remain intact in enterocytes untilincorporation into chylomicrons.

In embodiments according to the invention, the cholesteryl ester isobtained by esterifying cholesterol with a C₄ to C₃₆ saturated orunsaturated fatty acid, often a C₈ to C₂₆ fatty acid. In certainembodiments, the cholesteryl esters is often selected from the groupconsisting of cholesteryl myristate, cholesteryl laurate, cholesteryldodeconate, cholesteryl palmitate, cholesteryl arachidonate, cholesterylbehenate, cholesteryl linoleate, cholesteryl linolenate, cholesteryloleate and cholesteryl stearate.

Anti-Infective Molecules in Cholestosomes

In a preferred embodiment, the invention provides a cargo-loadedcholestosome pharmaceutical composition comprising an anti-infectivecompound (1) which is selected from the group consisting of miconazole,terconazole, econazole, isoconazole, tioconazole, bifonazole,clotrimazole, ketoconazole, butaconazole, itraconazole, oxiconazole,fenticonazole, nystain, naftifine, amphotericin B, zinoconazole andciclopiroxolamine, micafungin, caspofungin, anidulafungin, vancomycin,daptomycin, oritavancin, WAP 8294A, dalbavancin, ceftaroline, cefepime,ceftriaxone, ceftazidime, Quinupristin/Dalfopristin (synercid),fosfomycin, colistin, tigecycline and (2) which is encapsulated by asurface layer consisting essentially of a cholesteryl ester as otherwisedescribed herein. This composition can be used to treat an infection andcan be administered topically including orally or intravaginally.

Peptide Molecules Insulin and Beyond

In another preferred embodiment, the invention provides a cargo-loadedcholestosome pharmaceutical composition comprising a peptide which isoften selected from the group consisting of a hydrophilic peptide, humangrowth hormone, prolactin, oxytocin, calcitonin, bovine growth hormone,porcine growth hormone, Ghrelin, GLP-1, PYY36, Oxyntomodulin, GLP-2,Glucagon, and insulin, and which is encapsulated by a cholesteryl esteras otherwise described herein. This composition can be administered toincrease milk production, improve structure or function of organs andtissues such as pancreas or liver, to increase or initiate growth of amammal or to administer insulin in those individuals to whom insulintreatment is beneficial.

In certain embodiments, the surface layer of the nanoparticle is furtherenterically coated to prevent degradation of the pharmaceuticalcomposition in the gastrointestinal tract.

In certain embodiments, the surface layer of the cargo-loadedcholestosome remains intact at a pH range of between about 2 to about14.

In other embodiments, the cargo-loaded cholestosome is a unilamellarvesicle having a diameter of about 5 nm up to more than 10,000 nm (10micrometers), about 10 nm to about 1000 nm, often about 50 nm to about750 nm, about 100 to about 500 nm, about 200 to about 300 nm, dependingupon whether the material is subjected to an extrusion step or isunextruded. Accordingly, it is noted that larger cholestosomes are usedwhen the active molecule is larger and small cholestosomes are used whenthe active molecule is smaller.

Features of Oral Absorption and Favorable Associated Properties

While not being limited by way of theory, the present invention enablesoral delivery of a formulation that encapsulates a molecule into acholestosome which enters GI enterocytes through molecular recognition,is ingested, incorporates into a chylomicron, thereby fully protectingthe integrity of the molecule in the gastrointestinal tract, in theenterocyte, in the lymphatic system, in the blood, and across themembranes of body cells. Formulations of the invention do not release anactive ingredient until it has been taken into the cells of the body.Features of this invention thus include the following:

1) complete passage of Caco2 enterocyte barrier;

2) complete passage of cellular membrane barrier;

3) a method of intracellular delivery that largely avoids endosomeuptake;

4) oral delivery is independent of active molecule size, charge, bindingor degradation pathways, although the surface of the cargo-loadedcholestosome is itself neutral;

5) active ingredients circulate in lymphatics around the liver—an oraldelivery method that avoids first pass hepatic uptake; and

6) molecule delivery is facilitated by apolipoprotein attachments tosurfaces of chylomicrons, capable of docking with cells andintracellular loading, followed by unpacking of encapsulated moleculesin cytoplasm.

Accordingly, cargo-loaded cholestosomes according to the presentinvention are capable of delivering cargo (i.e., active molecules) to aconcentration within cells of a patient or subject to whom the presentcompositions are administered (preferably, orally) of at least 2 timesthat which is provided in the absence of administration in cholestosomes(i.e., by conventional pharmaceutical delivery means, including deliveryin liposomes). In most embodiments, the present invention deliversactive molecules within cells to a concentration at least 10 times, 25times, 50 times, 100 times, 250 times, 500 times and 1,000 times or morethat which is provided in the absence of cholestosomes. Thus, thepresent invention provides a means to encapsulate molecules of a varietyof size and molecular weight which heretofore could not be accommodated(itself an unexpected result) and regardless of size, the presentcompositions are capable of delivering active molecules to targets incells at concentrations much higher levels than the prior art.

These and other aspects of the invention are described in further detailin the Detailed Description of the Invention.

BRIEF DESCRIPTION OF THE FIGURES

Table 1 shows a comparison of properties between Cholestosomes andalternative delivery modalities evidences that cholesosomes are superioror at least equal in all categories. One particularly important aspectof this comparison is that nearly any molecule can be encapsulated intoa cholestosome without altering the molecule itself. This feature is notshared with other delivery systems, which tend to be specific to themolecule itself.

FIG. 1 shows diagrams comparing structural properties of chylomicrons,cholestosomes and liposomes, and assembly of a chylomicron containing acholestosome encapsulated molecule. The illustration shows the moleculewithin the cholestosome prior to the incorporation of the cholestosomeinto the chylomicron. The incorporation of Apolipoprotein (APO)structures into chylomicrons allows them to dock with cells and releasetheir contents, which include the cholestosome and its contents.Liposomes by contrast are a completely different physical structure,primarily composed of phospholipids and having a positively chargedsurface. Liposomes with positive charges are not taken into theenterocytes and thus they cannot enter into chylomicrons. Liposomes aretypically cleared primarily by the liver after they are injectedintravenously. Cholestosomes and chylomicrons bypass the liver vialymphatic channels, and that is the primary reason they loadencapsulated constructs into cells.

FIG. 2 shows a 3D Model of cholesteryl laurate/cholesteryl myristate ina 1:1 molar concentration ratio. A) bottom view slice B) top view slice.Red indicates negative charge (thin white arrows), blue indicatespositive charge (thick white arrow) and the yellow surface shows thetransition from one charged region to another (black arrows). The whitearrowhead in B indicates the predicted positions of the esterified fattyacid moieties. Notice that in these cutaway views, the surface can beseen as a region where docking could occur and depending on the natureof the esterified lipid, the cavity could have multiple separate sitesfor binding molecules.

FIG. 3 shows Gaussian distribution of cholestosome size in a preparationof not yet loaded cholestosomes, as measured using DLLS. Thecholestosomes in this preparation were 217+/−116 nm in diameter. Sizeranged from approximately 50 nm to approximately 500 nm. Scale is 1000nm which equals one micron

FIG. 4 shows sizing of cholestosomes during viewing of images fromtransmission electron microscopy. Cholestosomes were negatively stainedusing ammonium molybdate and imaged on a Hitachi H-500 transmissionelectron microscope. A scale bar is shown for estimation of size. Scaleunits of 1000 nm equals one micron equals one micrometer equals onemillionth of a meter. The median size range observed for cholestosomesin this microscopy, approximately 250 nm, is consistent with the sizedistribution determined by DLLS

FIG. 5 shows cholestosome-mediated delivery of FITC into MCF7 cells,measured after 24 hr of incubation. (A) Addition of FITC encapsulatingcholestosomes (ChF). (B) Addition of free FITC solution (FITC, 0.5M).(C) Addition of 100 uL of distilled water. Cells were incubatedovernight with ChF, FITC (100 uL) or vehicle (water) in complete medium.Cells were washed twice with medium and then incubated for 15 minutewith 10 uM Hoechst 33342 to stain nuclei. The later proves cellviability. Note the homogenous fluorescence in ChF treated cells (leftpanel in A), which indicates even distribution in cytoplasm.

FIG. 6 shows the impact of ester chain length during molecular modelingof cholesterol esters using SYBYL (Tripos, St Louis Mo.) on an HP XW8000workstation. Shown is the contrasting models formed using differentpairs of approximately equal alkyl chain length cholesterol esters. TheGASTIGER Huckel method was used to calculate charges, which are theinput for the electrostatic isopotential map. The isopotential surfacesdisplayed are at −10 and +10 kcal to highlight the ester link and thesterol nucleus. The red color is −10; blue is +10 kcals. The resultingdifferences in the center of the figures are the ester links. Note thatchanging the length of the esters in the model does not change thesurface or interior, but this change does bring the sterol nuclei closerto each other. It also changes size of internal diameter as well ascharacter of the hydrophobic “tracks”

FIG. 7 shows an image section of a matrix formed of cholesteryl estersof myristate and laurate. Charges were calculated and electrostaticpotential maps were generated. Note the blue/red regions, indicative ofa more hydrophilic region with the alkyl chains inter-digitating to formthe bilayer. The illustrated molecule is ceftaroline, a hydrophilicmolecule with mw approximately 600 daltons and size of 1.8 nm at itswidest point.

FIG. 8 shows Ceftaroline in relation to the matrix formed andillustrated by molecular modeling of cholesterol esters myristate andlaurate using SYBYL (Tripos, St Louis Mo.) on an HP XW8000 workstation.Interdigitizing alkyl chains are shown from the two cholesterol esters,in this example myristate and laurate. These structures form the outermembrane and the inner surface compatible with the molecule Ceftaroline,in this case. Ceftaroline is 1.8 nm at its widest spot. A cholestosomediameter is 250 nm. Based on membrane size, a fully loaded cholestosomeis 96% content inside with a water soluble molecule.

FIG. 9. shows a close in illustration of Ceftaroline in basiccholestosome matrix arranged in a ring. Ceftaroline is a very smallmolecule—1.8 nm and loaded in a 115 nm cholestosome, for relative sizeillustration purposes. The rings are chains of cholesteryl esters

FIG. 10 shows a partially assembled cholestosome around Ceftaroline,with one molecule shown. The partially assembled cholestosome matrixreveals walls and structures in relation to the ceftaroline molecule.Sizes include: membrane shown is 4 nm wide. Ceftaroline is 1.8 nm inlength. The internal diameter of the hydrophilic inner core pocket is 65nm

FIG. 11 shows molecular modeling of Insulin in relationship to thematrix of cholesterol esters using SYBYL (Tripos, St Louis Mo.) on an HPXW8000 workstation. Inter-digitizing alkyl chains are shown from the twocholesterol esters, in this example myristate and laurate. Thesestructures form the outer membrane and the inner surface compatible withthe molecule shown, in this case insulin. Sizes include: The membraneshown is 36 nm long and 4 nm wide. Insulin is 4 nm at widest spot. Acholestosome diameter is 250 nm. Based on membrane size, a fully loadedcholestosome hollow inner core could be as high as 96% of the contentinside, assuming a tightly packed center with a water soluble molecule.

FIG. 12 shows an image section of a matrix formed from myristate andlaurate assembled in an inter-digitated alternating alkyl chain modelaround Insulin. Charges were calculated and electrostatic potential mapswere generated. Note the blue/red regions indicate of a more hydrophilicregion with the alkyl chains inter-digitating to form the vesicle. Theillustrated molecule inside the insulin matrix (yellow) is ceftaroline,a hydrophilic molecule with mw approximately 600 daltons. The overlaywas performed to illustrate size differences, and does not imply anyintent by the inventors to combine these two molecules in the samecholestosome.

FIG. 13 shows a close in illustration of insulin in a basic cholestosomematrix, in this case formed from cholesterol esters of myristate andlaurate. Sizes include: the membrane shown as matrix is 36 nm long and 4nm wide Insulin is 4 nm at the widest spot. The diameter of thecholestosome vesicle shown is 100 nm for illustration purposes.

FIG. 14 shows molecular modeling of bevacizumab in relation tocholesterol esters using SYBYL (Tripos, St Louis Mo.) on an HP XW8000workstation. Interdigitizing alkyl chains are shown from the twocholesterol esters, in this example myristate and laurate. Thesestructures form the outer membrane and the inner surface compatible withthe molecule shown, in this case bevacizumab. Sizes include: bevacizumabat 17 nm long and 4 nm wide, while the membrane shown is 36 nm long and4 nm wide in a single matrix ring. A cholestosome diameter forBevacizumab unextruded is 10,000 nm and extruded is 250 nm. Based onmembrane size, a fully loaded cholestosome bound with a single bilayerring has 96% of the content by weight to weight inside with a watersoluble molecule.

FIG. 15 shows a close in illustration of Bevacizumab in a basiccholestosome matrix, in this case formed from cholesterol esters ofmyristate and laurate. Volume of a 250 nm cholestosome is 7 millioncubic nanometers, or 7×10⁻¹⁵ ml. Assuming it is unilamellar, if we arecalculating making a loading factor out of this the cholestosomecontains 4% of the volume and the solution within it contains is 96% ofthe volume. Sizes include: bevacizumab at 17 nm long and 4 nm wide; themembrane shown has a diameter of 100 nm

FIG. 16 shows molecular modeling of Bevacizumab, Insulin, Ceftaroline ina basic Cholestosome matrix formed from cholesterol esters using SYBYL(Tripos, St Louis Mo.) on an HP XW8000 workstation. Interdigitizingalkyl chains are shown from the two cholesterol esters, in this examplemyristate and laurate. These structures form the outer matrix and theinner surface compatible with the molecules shown, in this casebevacizumab, insulin and ceftaroline. Based on membrane size, a fullyloaded cholestosome is 96% content inside when loaded with a watersoluble molecule. Loading can be calculated based on v/v (volume of thevesicle and volume of the molecule). Assumptions: the cholestosome is asphere with a diameter of 250 nm. The internal core hydrophilic pocketdiameter is then 242 nm with a radius of 121 nm. This results in a totalcholestosome volume of approximately 7 million nm3 (cubic nanometers).We can calculate the volume of a molecule and calculate the totalnumbers potential (not including solvent) and calculate a mass ratio ofvesicle to molecule in that manner. Another way is to consider thevolume of the vesicle and consider the concentration of the analog to beencapsulated. For example, bevacizumab in solution at 100 mg/ml.Converting cubic nanometers to ml and then determine how much can fit ina cholestosome and compare mass in that manner. Upon conversions, thisresults in a mass ratio where bevacizumab to lipid ratio in content isabout 96:4%

FIG. 17 shows an illustration of the apparatus used to collectbasolateral fluids following exposure of the apical side of a monolayerof Caco2 cells to a cholestosome encapsulated molecule. Cholestosomeencapsulated molecules of all sizes are taken into Caco-2 cells, andfrom there the loaded cholestosomes are incorporated intact intochylomicrons by the Golgi apparatus. The uptake process by enterocytesis more rapid and efficient than the process shown here for Caco-2cells. Other typical components of Chylomicrons are APO-B, otherapolipoproteins, and triglycerides. After formation, chylomicrons aresecreted by Caco-2 cells into the lymphatic fluid on the basolateralside of the monolayer. Chylomicrons loaded with cholestosomes arecaptured in the fluid on the basolateral side of the Caco2 monolayer.

FIG. 18 shows an apical side placement of FITC insulin placed for 1 hour(not in cholestosomes) on Caco-2 cells. Image was taken at 1 hr byfluorescence microscopy, and the image here was taken of both sides ofthe entire cell system, Green is FITC label. Without a doubt the FITCsignal stays on the apical layer in the main. However, there are signsthat the FITC insulin (or the FITC fragments in the case of digestedinsulin) may be taken into the Caco-2 cells; note the aggregationparticles (e. g. arrows). It could conceivably be chylomicron uptake ofFITC or fragments of FITC insulin that create the large fluorescentstructure at the bottom right (arrowhead).

FIG. 19 shows the results of a transwell experiment, with an image takento show baseline conditions with image of fluid at the basolateral side.Nothing was applied to the apical layer of the Caco2s (PBS/glucoseonly), PBS only in basolateral chamber, so the image reflects nativefluorescence of the Caco-2 cell system used for our testing. Fluid wastaken from the basolateral side and then imaged at 200× power. Scale is10 um, which is 10,000 nm

FIG. 20 shows the results of a transwell experiment with FITCcholestosmes, exactly the same conditions as previously; This time theinventors applied cholestosome with FITC in PBS to the apical side andleft on for 2 hr. Based on sizing of these imaged chylomicrons, (20-30um=27,000 nanometers (which is 100× larger than a FITC cholestosomeapplied to the apical side). The inventors conclude these are likely midto large sized chylomicrons that have incorporated many of the 250 nmFITC-cholestosomes inside. Note that these images are made on only thefluid from the basolateral side, NOT imaging the prep before removingthe fluid. Image here is 200× magnification. There is likely to be freeFITC-cholestosomes remaining in the solution applied to the apical side,but this cannot be quantified here because this image shows just thebasolateral fluid after removal from the transwell preparation.

FIG. 21 shows the results of a transwell experiment, exactly the sameconditions as previously, this time the inventors appliedcholestosome-FITC-insulin in PBS and left on for 2 hr. Based on howsizing of the chylomicron images (at 40-60 um, which is 40,000-60,000nanometers), these are relatively large chylomicrons that haveincorporated FITC-insulin-cholestosomes inside. Note that the inventorsare imaging only the fluid from the basolateral side. Image here istaken at 200×, the scale shows 10,000 nm

FIG. 22 shows the original starting concentration for FITC-insulinexposure in MCF-7 cells was 466 mcg/ml, which did not result inmeasurable amounts of FITC insulin inside the MCF-7 cells in row A. Forthe two lower figures (rows B and C), the concentration of FITC insulincholestosome was 0.46 mcg/ml, which is the same for the experimentssummarized in the last 2 figures. The 0.46 mcg/ml from FITC insulincholestosomes (row B) produced about the same intracellular fluorescenceas 466 mcg/ml of FITC insulin without cholestosomes (row A). Compared to466 mcg/ml of FITC insulin without cholestosomes (row A), the furtherprocessing of FITC insulin cholestosomes by Caco-2 cells intochylomicrons, produced a robust improvement in the amount of insulininside cells from FITC insulin cholestosome-chylomicrons (row C), muchgreater than 1000 fold over the amount of FITC-insulin alone, and muchgreater than the effect of the 0.46 mcg/ml of insulin when not processedby Caco-2 cells. Assuming the amount passing Caco2 cells was all of theinsulin administered to the apical side, the concentration of insulin inthe FITC insulin cholestosome chylomicron row C was the same as theinsulin concentration in the middle row Row B. This particularpreparation had free insulin remaining, and if transfer across Caco-2cells was less than 100%, then these intracellular loading ratios areeven greater. Clearly, FITC insulin cholestosome-chylomicrons achievesgreater loading inside the cells, once again demonstrating thatcholestosomes alone do allow peptides to enter cells across the cellmembrane, as was earlier shown with FITC cholestosomes alone. The imagein the bottom row C reflects the observed penetration of FITC insulincholestosome chylomicrons inside cells. Not only are the cell membranesdramatically more concentrating FITC insulin in this image, but thecytoplasm of these cells is loaded with FITC insulin as well. This isafter only 2 hr exposure, confirming that chylomicrons not only loadmassively more, they load more quickly than cholestosomes on their own.

FIG. 23 shows a comparison of MCF-7 cells exposed to preparations ofFITC tobramycin by bright field vs FITC fluorescence imaging shows 1) anoverall successful loading of MCF-7 cells after 24 hr exposure toFITC-cholestosomes, which has been shown repeatedly in our work withcholestosomes. In 2), this response, essentially no effect from anexternal concentration of 700 mcg/ml, is compared with the general lackof intracellular loading of MCF-7 cells when exposed to FITC-tobramycin.This is expected because tobramycin does not enter most body cells, andany cell that takes up tobramycin actively is subject to theintracellular killing from tobramycin. This is the basis for tobramycinnephro and oto toxicity. In 3) and of great interest, when MCF-7 cellswere exposed to FITC-Tobramycin-cholestosomes for 24 hr, these MCF-7cells all died, as can be seen in the last frame at both top and bottom.The purpose here is to show how tobramycin, when it enters cells, is ageneral toxin to the mitochondria and when tobramycin enters even cellsotherwise resistant to its intracellular effects, there is potential forintracellular uptake and harm.

FIG. 24. Vancomycin entry into MCF-7 cells at 24 hr. In this series ofexperiments, the original starting concentrations of vancomycin werebetween 41 and 666 mcg/ml. In each column, the top image is thefluorescence, the bottom is the darkfield. Displayed out of thisFITC-vancomycin series in column B is FITC vancomycin at 83 mcg/ml. Incolumn A, FITC-vancomycin-cholestosomes at 0.83 mcg/ml produced greateruptake at a value 100 fold lower than the vancomycin concentration inFITC-vancomycin column B. The fluorescence image in column A shows moreloading than the image in column B, indicating that the MCF-7 cellularloading ratio is more than 100× greater withFITC-vancomycin-cholestosomes. When the concentration of FITC-vancomycinwas increased to 666 mcg/ml in column C, these cells are still notloading as high as those in column A. The fluorescence data on loadingof FITC vancomycin is therefore approaching 1000× greater whencholestosomes are used. It should be noted that there was no effect ofhigh amounts of FITC vancomycin cholestosomes on these MCF-7 cells. Theimages in the three panels confirm our observed penetration of FITCvancomycin cholestosomes inside cells. Not only are the cell membranesdramatically more concentrating FITC vancomycin in this image, but thecytoplasm of these cells is loaded with FITC vancomycin as well. This isafter only 24 hr exposure, confirming that cholestosomes load massivelymore vancomycin in the cells.

FIG. 25 shows that FITC insulin cholestosome loading of MCF-7 cells wasimproved over some of our previous experiments with FITC insulincholestosomes, and here the loading was even greater from FITC insulincholestosome chylomicrons. In all cases, processing of FITC insulincholestosomes by Caco-2 cells into chylomicrons, produces a robustimprovement in the amount of insulin inside cells from FITC insulincholestosome-chylomicrons (row B), Not only are the cell membranesdramatically more concentrating FITC insulin in this image, but thecytoplasm of these cells is loaded with FITC insulin as well. This isafter only 2 hr exposure, confirming that chylomicrons not only loadmassively more, they load more quickly than cholestosomes on their own.This formulation was administered to 4 mice.

FIG. 26. Four mice were given the FITC insulin cholestosome formulationorally, with subsequent 30 minute blood glucose measurements using aglucometer. Three of the four mice dropped blood glucose substantiallybetween 30 and 60 minutes after oral gavage with the FITC insulincholestosome preparation. The fourth mouse did not drop blood glucoseuntil 2 hr after administration, but had a similar decline and recoverytime. Data are shown in this figure both individually and together.

FIG. 27 shows dark field (top row) and fluorescent images at 2 hr, 4 hrand 24 hr from the application of a target concentration of 173 mcg/mlof FITC Bevacizumab to MCF-7 cells. These concentrations are 5-10 foldgreater than typically observed in Bevacizumab treated patients. Therewas no evidence that FITC Bevacizumab integrated into the MCF-7 cellmembranes of these MCF-7 cells. There was no evidence of anyfluorescence uptake of FITC bevacizumab at any time point by MCF-7cells, and there was no evidence of effect of FITC-Bevacizumab on theseMCF-7 cells. The IC50 for Bevacizumab against MCF-7 cells isapproximately 1.0 mcg/ml.

FIG. 28 shows FITC bevacizumab cholestosomes and FITC-bevacizumabcholestosome chylomicrons which were prepared and tested against MCF-7cells. There was no effect at 2 hr, at which point the MCF-7 cellsshowed little uptake of FITC bevacizumab cholestosomes. As these sameFITC bevacizumab cholestosomes were placed on the apical side of theCaco-2 cells and the resulting FITC bevacizumab cholestosomechylomicrons were collected, these FITC bevacizumab cholestosomechylomicrons were tested on MCF-7 cells. The first frame of the bottomrow shows massive uptake of FITC bevacizumab cholestosome chylomicrons,and as shown in the next frames, all the MCF-7 cells were killed by 4hrs into the experiment. This was completely unexpected based on theknown mechanism of action of Bevacizumab

FIG. 29. Shows the assembly of a lipid nanoparticle from cholesterylmyristate, cholesteryl laurate and Insulin in the hollow core.

DETAILED DESCRIPTION OF THE INVENTION Cholestosornes are Unique andNovel Over any Prior Art

Cholestosomes pursuant to the present invention are unique amongdelivery systems for molecules. Unique among drug delivery means, theinventors have successfully disguised proteins and other molecules andchemical compounds as components commonly known in the art as food. Mostspecifically, the chosen materials for oral uptake are dietarycholesteryl esters. Surprisingly the cholesteryl esters provide a uniquecholesteryl ester nanoparticle having the following properties thatdifferentiate cholestosome encapsulated products (especiallymacromolecules which cannot otherwise be delivered to patients with anyreal measure of success) over liposomes or any other nanoparticle:

-   -   1. All component materials of the delivery means and system are        common dietary ingredients, and total dosage of these substances        per day in most applications will be less than from food.    -   2. Working temperature for encapsulation in cholestosomes is        often 35-45 degrees centigrade, which is an optimal temperature        for the stability of peptides and proteins in their body        circulating forms.    -   3. Said Delivery means will offer all favorable aspects without        concern for molecular size, charge, binding or degradation        pathways    -   4. Cholestosome encapsulated proteins show complete passage of        Caco2 enterocyte barrier, and are incorporated intact into        chylomicrons    -   5. Bypass of the liver and associated first pass clearance        pathways    -   6. Cholestosomes and the chylomicrons that contain them, provide        protection for molecules as they pass cell membranes from oral        intake all the way to intracellular uptake    -   7. Docking with cells; Quantitative intracellular loading;        Complete passage of cellular membrane barrier    -   8. Unpacking of encapsulated contents in cytoplasm by        cholesteryl ester hydrolases, an endogenous pathway.    -   9. Robust intracellular concentration of payload molecules at        intracellular sites, yet cholestosomes do not use endosome        uptake pathways    -   10. Cholestosomes and their encapsulated contents are        distributed into all cells when incorporated into native formed        chylomicrons

While some deliver systems may achieve one or a small number of these 9features, there is no other delivery system that can achieve this widearray of favorable properties, especially when the delivery systemenables oral use of heretofore unabsorbed proteins, and does not alterthe payload molecules and can be employed for essentially any moleculeor chemical compound. Cholestosomes are the first intracellular deliverysystem that can be applied to any molecule. In fact, cholestosomes areat least as efficient with macromolecules, especially includingproteins, peptides, polynucleotides (RNA and DNA, including, forexample, naked DNA, plasmid DNA, interfering RNA or “RNAi”, includingsmall interfering RNA or “siRNA”, small hairpin “shRNA”, bifunctionalshRNA, microRNA and various oligonucleotides of DNA and RNA, amongothers) and macromolecular antibiotics, among others, as they are withsmall molecules.

Because of the unique mechanism of delivering active molecules to atarget within cells of a patient or subject, cargo-loaded cholestosomesaccording to the present invention are capable of delivering cargo(i.e., active molecules) within cells of a patient or subject to whomthe present compositions are administered (preferably, orally) to aconcentration of at least 2 times that which is provided in the absenceof cholestosomes (i.e., by conventional pharmaceutical delivery means,including delivery in liposomes). In most embodiments, the presentinvention delivers active molecules within cells to a concentration atleast 10 times, 25 times, 50 times, 100 times, 250 times, 500 times and1,000 times or more that which is provided (delivered into cells) in theabsence of cholestosomes.

As cholestosomes are novel in relation to prior art for molecule andchemical compound delivery, the inventors provide detailed comparisoninformation to the reader in order to point out why prior art does notdisclose any similar system;

Following these comparisons, Non-limiting examples will be provided.

The following terms are used throughout the specification to describethe present invention. Where a term is not given a specific definitionherein, that term is to be given the same meaning as understood by thoseof ordinary skill in the art. The definitions given to the diseasestates or conditions which may be treated using one or more of the lipidnanoparticle encapsulated compounds according to the present inventionare those which are generally known in the art.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “a compound” includes two or more different compounds. Asused herein, the term “include” and its grammatical variants areintended to be non-limiting, such that recitation of items in a list isnot to the exclusion of other like items that can be substituted orother items that can be added to the listed items.

Cholesterol has vital structural roles in membranes and in lipidmetabolism in general. It is a biosynthetic precursor of bile acids,vitamin D and steroid hormones (glucocorticoids, oestrogens,progesterones, androgens and aldosterone). In addition, it contributesto the development and working of the central nervous system, and it hasmajor functions in signal transduction and sperm development. It isfound in covalent linkage to specific membrane proteins or proteolipids(hedgehog′ proteins), which have vital functions in embryonicdevelopment.

Cholesterol esters, preferably with long-chain fatty acids linked to thehydroxyl group (often prepared from fatty acids containing at leasteight up to 26 carbon atoms), are much less polar than free cholesteroland appear to be the preferred form for transport in plasma and as abiologically inert storage (de-toxification) form. They do notcontribute to biological membranes but are packed into intracellularlipid particles.

Cholesterol ester hydrolases in animals liberate cholesterol and freefatty acids from the ester form, when required for membrane andlipoprotein formation. They also provide cholesterol for hormonesynthesis in adrenal cells. Many cholesterol ester hydrolases have beenidentified, including a carboxyl ester hydrolase, a lysosomal acidcholesterol ester lipase, hormone-sensitive lipase and hepatic cytosoliccholesterol ester hydrolase. These are located in many different tissuesand organelles and have multiple functions.

The applicants disclose a novel delivery technology which encapsulatesmolecules in a cholesteryl ester particle called a cholestosome, andafter this particle is orally absorbed by cells of the intestine, it isplaced into chylomicrons for delivery to all body cells via lymphatictransport. After this nanoparticle is taken up into cells from thechylomicron transport particle, the cholesterol ester hydrolases unpackthe particle and liberate the molecule at the intracellular site.

Relevant background information regarding the structure of thecholestosomes in this application is found in United States PatentApplication Document No. 20070225264, filed Mar. 20, 2007 and entitled“Drug Delivery Means”.

Principles of interdigitation as used herein are known to those ofordinary skill in the art. See e.g. Yeagle, The Structure of BiologicalMembranes (CRC Press 2010).

“Chylomicrons” are very large, heterogeneous, lipid-rich particlesranging in diameter from about 750 to 40,000 nm. They are formed in theenterocytes of the GI tract and function to transport dietary fat andfat-soluble vitamins to cells via circulating in the bloodstream. Adiagram of the formation of chylomicrons from cholestosomes and otherlipid particles is shown as FIG. 1. The size heterogeneity of thesecreted chylomicron particles depends on the rate of fat absorption,type and amount of fat absorbed. When cholestosomes are very large, theresulting chylomicrons that incorporate these large cholestosomes can belarger as well.

“Cholestosomes” are stable in the adverse conditions of the GI tract,possess greater design flexibility, and exhibit greater encapsulationefficiency for a wide variety of molecules, and have advantages ofeasier manufacturability. These favorable cholestosome properties areemphasized in Table 1, which compares delivery systems. The structuraldifferences between cholestosomes and liposomes confer on cholestosomesdifferent physical and chemical properties and therefore permit themsuperiority in desired properties and functions. For example,cholestosomes have been shown to be stable over a wide pH range from 2to 13. In contrast, according to a 2005 review article in the Journal ofMolecular structure describing liposomes, “owing to the small resistanceof liposomes to gastric juice (pH 1.9), enzymes of the alimentary canaland bile acids in the intestine (pH 8) their application per os isuseless.” Cholestosomes resist pH degradation and therefore have thepotential to be used as a primary means for oral delivery of molecules,a particularly novel aspect of the present invention.

A “cargo-loaded cholestosome” refers to a cholestosome which hasencapsulated a pharmaceutically active agent and contains the agentprincipally, although not necessarily exclusively, in the core of thecholestosome vesicle.

Secondly, the structural features based on the interaction of thecholesteryl esters confers electrostatic surface properties which arecalculated to be similar to PEG surfaces which liposomes use to conferenhanced time in the blood system. This confers upon the drug ormolecule contained within the cholestosome a longer residence time inthe body, normally an advantage of a drug delivery system, but notnecessarily an advantage if the molecule cannot be released from thedrug delivery nanoparticle.

The evidence for this is the Zeta potential measurements showingcholestosomes with a neutral surface in one formulation cholestosomeshave a measured Zeta potential of −14, which is typical of a neutralcharge to cholestosomes alone in their unloaded form. Neutral chargeboundaries for Zeta potential means having a Zeta potential of about −20to about +20, often about −40 to +10, −5 to +5 or approximately 0. Thepush for neutral surface charge leads to the use of PEG is used in othertypes of formulations. Cholestosomes approximate the neutral surfaces ofPEG in certain comparisons of embodiments among the various inventions.

Structural modifications of cholestosomes are based on modification ofmole ratios of the esters which result in different interior andexterior surface properties and in cholestosomes those properties arenot defined by an organization based on hydrophilic/hydrophobicsequestration (as in liposomes and other prior art delivery means) andtherefore are more easily defined and manufactured. (Evidence of size asa result of sonication, often temperature, often pH (aqueous solutionsof neutral pH have different charges on the molecules for encapsulationwhich may affect their ability to define the size of the lipidnanoparticles)). All of these beneficial properties are summarized andcompared with those of other delivery systems in Table 1 below.

As shown in Table 1, a comparison of properties between Cholestosomesand alternative delivery modalities evidences that cholesosomes aresuperior or at least equal in all categories. One particularly importantaspect of this comparison is that nearly any molecule can beencapsulated into a cholestosome without altering the molecule itself.This feature is not shared with other delivery systems, which tend to bespecific to the molecule itself. Design flexibility is an advantageousproperty for a drug delivery system, clearly evident in the presentinvention.

TABLE 1 Comparison of properties of Cholestosomes with other commonmeans of drug delivery Table 1. Comparison of properties betweenCholestosomes and alternative delivery modalities. Synthetic CarrierCholestosome ™ Polymers Liposome Proteins Stability in + + + +Bloodstream Survivability in GI +++ + + + tract Design Flexibility +++ ++/− − Manufacturability +++ + ++ + Efficacy + + + + Encapsulation+++ + + + Efficiency

In Table 1 above, synthetic polymers refers generally to techniques suchas PEGylation. Carrier proteins refers to attached biological moleculessuch as viral vectors. Both PEGylation and Carrier proteins constructsare given intravenously, and like liposomes, are not absorbed if givenorally, primarily because they are degraded in the GI tract

Loading Properties of Cholestosomes Vs Liposomes

Liposomes rarely load even 1% weight:weight even when using a lipophilicmolecule such as doxorubicin. Cholestosomes as developed by theinventors often will load at least 20% and theoretically much higher,weight:weight as otherwise described herein.

Liposomes do not Load Proteins but Cholestosomes Load themPreferentially

Liposomes do not load proteins, genetic materials (polynucleotides, suchas DNA and/or RNA as otherwise described herein), peptides (especiallyincluding polypeptides such as monoclonal antibodies) and manymacromolecules including macromolecular antibiotics in usable amounts(less than 2% means that the amount of carrier is very large ifencapsulating a dose of 100-1000 mg which is typical of peptides ormonoclonal antibodies). Many molecules which are water soluble, andwhere the charge is positive, are not favorably loaded intonanoparticles like phospholipid based liposomes. In contrast, the insideof a cholestosome (core) is large in relation to the size of theencapsulating membrane, and hydrophilic but neutral, a system compatiblewith loading proteins, peptides, genes as well as hydrophilic smallmolecules which are charged. Since all of these fail to pass the GItract barrier, the use of Cholestosomes offer, for the first time, theprospect of orally absorbed proteins and peptides.

Neutral Charge of Cholestosomes Vs Positive Charge of Liposomes

Liposomes are not able to pass the Caco-2 enterocyte barrier intact, infact most are broken open in the GI tract to harvest their individualcomponent phospholipids. Thus liposomes and their payloads are not takenup by enterocytes, perhaps due to their surface charge. Cholestosomesare comprised of Cholesteryl esters, which are already converted bycholesterol esterases into absorbable moieties. They are already neutralparticles by virtue of their composition from cholesteryl esters, andare preferred in this form by the enterocyte cells of the duodenum forabsorption intact and use in chylomicron formation. As long as theencapsulated molecule is completely within the hollow center,cholestosomes are taken up intact and they are placed intact intochylomicrons in the golgi apparatus of enterocytes.

Liposomes do not Pass Cell Membranes

Not only do liposomes fail to be orally absorbed with their payloads,they also do not enter cells and certainly when lacking APO on theirsurfaces, they have no ability to dock with cells in need of lipids.When injected intravenously, Liposomes are harvested by the liver andthere broken down into their component phospholipids. This does notordinarily offer intracellular delivery of their contents, although highlocal concentrations of payload molecules in the liver may offer anadvantage if the target cell is the hepatocyte.

Liposomes and Therefore their Contents do not Enter Chylomicrons

Phospholipid coatings of liposomes are degraded in the GI tract, andthus the liposome itself has been degraded and its contents released inthe GI tract, and even before arrival at the duodenal site ofabsorption. Thus even if a protein could be loaded into a liposome, itwould be destroyed with the liposome before it could be absorbed byenterocytes. There is no chance for a phospholipid constituent liposometo be incorporated into a chylomicron.

Cholestosomes do not Enter Cells on their Own

Intravenously administered, cholestosomes would not dock with cells, asthey are lacking the surface apolipoproteins which are necessary fordocking with the cells. However, cell membranes do appear to trapcholestosomes, and from this vantage point the parenteral use ofcholestosomes does allow some intracellular uptake of certain molecules.Intracellular uptake is much greater if these same cholestosomes aregiven orally. Topical administration, including vaginal administrationis also preferred.

Cholestosomes clearly enable greater amounts cell uptake after oralabsorption because they are first taken into chylomicrons. Chylomicronsthen selectively deliver lipids to cells which are in need thereof.Cells in need express a docking site protein which then can link to theAPO-B on the surface of the chylomicron, thus effecting docking andrelease from the chylomicron into the cytoplasm of the cell Furthermore,the chylomicrons that are formed from cholestosomes have Apolipoproteinrecognition properties on the surface that reaches every cell. Aschylomicrons contact cells, they dock with cells that are expressingsurface proteins and thereby requesting transport of lipids includingtriglycerides and cholesteryl esters. After lipases are disgorged fromthe cell, said lipids such as triglycerides and the cholestosomes aretaken into the cell including their encapsulated payloads.

By Contrast, when liposomes are injected into the blood they would notbe expected to dock with cells, as they are lacking Apo E constituentsfor docking with cells seeking lipids. Liposomes serve to create aprolonged plasma release characteristic to molecules in drug delivery.Furthermore, in the favorable occasion where the drug encapsulatedwithin a liposome delayed release system does enter the cell, then itwould be expected that there is intracellular delivery of payloadbecause of the property of the drug after it is freed from the carryingliposome.

Formulation of Cargo-Loaded Cholestosomes—One of ordinary skill in theart can readily look at any molecule and predict which cholesterylester(s) should be used to form cholestosomes

Cholestosomes are formed in several stages, first by dissolution of thepair of chosen cholesteryl esters in organic solvent such as ether, thenremoval of the organic solvent, and next there is addition of aqueouscomponent which contains the molecule to be encapsulated, withsonication to form the unilamellar membranes and generate thehydrophilic relatively uncharged hollow pocket around molecules inaqueous.

All formation stages are carried out in a water bath at a criticalspecified temperature which is based on the lowest melt temperature ofthe esters. Working temperature is a primary condition for selection ofcholesteryl ester pairs, as the melt temperature of the chosen pairs ofesters must be equal to or lower than the temperature that will degradethe molecule chosen for encapsulation. With the temperature limits inmind, the cholesteryl ester pairs must be chosen to form a bilayermembrane at temperatures below 40 C, which is a basis for choice ofcholesteryl myristate and cholesteryl laurate for many of the examplesof encapsulated monoclonal antibodies in this disclosed invention.

FIG. 2 depicts a three dimensional model of a cholesteryllaurate/cholesteryl myristate (1:1 molar concentration) cholestosome.Cholestosomes can have a wide range of sizes, as shown in FIGS. 3 and 4.Active ingredient load can be determined through calculations such asthose shown in the legends of FIGS. 15 and 16.

Once there is the addition of the aqueous molecule or construct, themixture is sonicated until there is a cloudy solution formed, therebyminimizing waste from un-dissolved esters, with sonication providingenergy for unilamellar vesicle formation. The aqueous component is alsomaintained at the target temperature prior to its addition, and asstated previously for most peptides, proteins and genes, the highesttemperature that can be tolerated is only about 40° C.

The solution is then filtered and the filtrate is saved for extrusionfor size conformity. The sample is then stored in the refrigerator.

The newly encapsulated molecule is surrounded by the unilamellarcholesteryl ester vesicle and inside the hollow pocket the encapsulatedmolecule is protected from contact with the harsh environment of the GItract and is held away from enzymes and the cells of the immune system.The molecule inside remains unchanged. Accordingly, providingcargo-loaded cholestosomes pursuant to the present invention is afacile, routine undertaking.

Cholesteryl Ester Chain Length

The outer membrane of Cholestosomes consists of cholesteryl estersarranged to form a lipid nanoparticle based upon cholesteryl esters,generally in the case where the plurality of cholesteryl esters have thesame or similar molecular length, so as to form a uniform capsule arounda macromolecule encapsulated by said cholestosome. The cholesterylesters may be of different lengths as long as they are co-soluble, whichwill permit them to aggregate together to form a vesicle with a ratherlarge hollow core in relation to the total size of the lipidnanoparticle. In fact, some configurations have the core displacementwell beyond 80 percent of the entire nanoparticle, which affordsbeneficial high loading of water soluble molecules such as insulin.

This is based on the ability of differential mole fractions of differentesters being able to co-exist and aggregate in a minimum energyconformation in which the vesicle formation is determined by the natureof the cholesteryl esters and their relative mole fractions.

An illustration of an assembling nanoparticle around a molecule, in thiscase insulin is found in FIG. 29. Here, the chains are configured in acircular format so as to form a hollow center which has a neutral ormildly negative charge (Zeta potential measurements are made to definethis property, as will be shown in the examples for each formulationdisclosed. Cholestosomes alone have a Zeta Potential reading of −14).

With Insulin in the cholestosome, its Zeta Potential goes additionallynegative. Cholestosome encapsulated formulations do not have highlypositive charges, in contrast to Liposomes, where the Zeta potentialcould range as high as +76 in some experiments.

In this and other examples, the cholesteryl esters may be of differentlengths as long as they are co-soluble, which will permit them toaggregate together to form a unilamellar vesicle. This is based on theability of differential mole fractions of different esters being able toco-exist and aggregate in a minimum energy conformation in which thehollow core of the nanoparticle is determined by the nature of thecholesteryl esters and their relative mole fractions.

As the assembly of cholestosomes are considered and with reference tothe 3D diagram as FIG. 2, consider first the assumption that theinterior and exterior of the cholestosome matrix are the samestructurally in that the sterol nuceli point both into the cavity andout to the surface.

What can be changed by choice of cholesteryl esters is the length of theester tails. Having a shorter tail length brings the sterol nucleicloser to each other and lessens the hydrophobic nature of the vesicle(due to chain length). This may have an enhancing impact on thehydrophilic character of the cholestosome. This can be modeled andexamples are presented in FIGS. 6 and 7.

Furthermore, assuming the same packing of the inner core of the lipidnanoparticle irrespective of chain length, shorter chains around alarger molecule would increase the mass to mass ratio of the molecule tothe lipid. Clearly, larger molecules need larger internal cores, andhence ester chain length is important for the construction of largercholestosomes to accommodate larger molecules such as monoclonalantibodies.

Balanced against these considerations is the impact of ester chainlength on the relative hydrophilicity of the inner core. Longer esterchains increase the hydrophobic character and allow for packing of amore hydrophobic molecules into the core.

There is also the issue of interactions of matrix cholesteryl estersinteracting with solvents. Aqueous solvent combinations includingethanol may help in the encapsulation process overall, and increase theamount encapsulated at a fixed ratio of cholesteryl esters. This is theimpact of charge of the construct and charge of the inner core of thecholestosome.

For example, in crystal structures of oxysterols, changing the solventratio by including an alcohol such as ethanol helps bring the oxygenmolecules closer to each other, which may help the esters orient in acholestosome and also help bring the molecules into the core of thecholestosome vesicle more readily.

In modeling of these interactions, the inventors can examine models ofcholesteryl ester matrix structures and predict which esters are thebest choices for specific molecules or drugs. A systematic approach ispossible when the interactions between esters, charge, solvents andmolecule are considered simultaneously.

Effect of Particle Size on Target

Cholestosome component mixtures differ in novel ways depending on theionic and physicochemical characteristics of the macromolecularcomponent. The size of the cargo-loaded cholestosome often affects thetarget in that certain cholesterol esters, when formed intocholestosomes, are better suited for delivering certain molecules andthus the impact of ester chain length,

How the Charge and Molecular Pattern Impact the Size of the Cholestosome

Larger molecules with greater net positive charges need longer chainlength cholesteryl esters for optimal encapsulation, provided the melttemperature is compatible with the stability of the molecule beingencapsulated, throughout the encapsulation process. Smaller moleculeswith a lower net positive charge may be encapsulated with shorter chainlength cholesteryl esters. Adjustment of the cholesteryl ester chainlength to provide lipid nanoparticles based upon cholesteryl esterspursuant to the present invention is well within routine skill.

Surfaces on said cholestosomes may either be smooth, or rough, dependenton component balance and mixture characteristics. The character of thevesicle surface will depend on the esters themselves as well as theinteraction of the esters with each other. The expectation is that theesters will aggregate to optimize the molecular interactions and tominimize the holes or spaces between them. These arrangements maytherefore produce a surface that is rough.

Most of the graphical examples in the figures of this disclosure haverough configuration, as the esters have arranged themselves so thatstructural components are inter-digitated on the vesicle surface toproduce an uneven structural arrangement (rough). In some cases theesters have arranged themselves so that they are aligned to produce asurface of constant shape and size (smooth).

The nature of the final surface configuration will depend on thecombinations of esters used and their relative concentration in theformulation. In summary, both the choice of esters and the choice ofmolecule affect the final arrangement of the lipid nanoparticle. Whilethe various components affect the surface configurations, a novelsurface property, the neutral surface itself that allows for uptake byenterocytes, should be the net effect of the charges of the chosenmolecules in the final formulation. The surface should always be neutral

Properties of Formed Cholestosomes and Illustrated Examples

Larger molecules with greater net positive charges need longer chainlength cholesteryl esters for optimal encapsulation.

Preferred cholesteryl esters for use with larger water solublemacromolecules such as proteins and peptides are those which areprepared by the esterification (or a related process to provide thecorresponding cholesteryl ester) of a C₈ to C₂₆ saturated or unsaturatedfatty acid, often a fatty acid selected from the group consisting ofMyristoleic acid, Palmitoleic acid, Sapienic acid, Oleic acid, Elaidicacid, Vaccenic acid, Linoleic acid, Linoelaidic acid, α-Linolenic acid,Arachidonic acid, Eicosapentaenoic acid, Erucic acid, Docosahexaenoicacid, Caprylic acid, Capric acid, Lauric acid, Myristic acid, Palmiticacid, Stearic acid, Arachidic acid, Behenic acid, Lignoceric acid,Cerotic acid or a mixture thereof.

The mixing of more than one (preferably two) cholesteryl ester to formcholestosomes may accommodate different sized active molecules withvarying delivery characteristics.

FIG. 2 depicts a three dimensional model of a cholesteryllaurate/cholesteryl myristate (1:1 molar concentration) cholestosome.Cholestosomes can have a wide range of sizes, as shown in FIGS. 3 and 4.Active ingredient load can be determined through calculations such asthose shown in the legends of FIGS. 15 and 16.

A discussion of the prior art delivery methods that have been attemptedwithout success for proteins and peptides has been presented, allowingthe skilled practitioner to understand that prior delivery methods donot possess most of the critical embodiments necessary for successfuloral use of proteins in human patients. From this disclosure also, it isclear to those skilled in the art how Cholestosomes solve each and allof these prior deficiencies with respect to oral absorption andsubsequent delivery to body cells after incorporation into chylomicrons.

For example, FIG. 5 depicts cholestosome-mediated delivery of FITC intoMCF7 cancer cells, evidencing the invention's ability to deliver activeingredients to therapeutic sites that necessitate precise activeingredient targeting. Subsequent examples with show more cholestosomepreparations that pass cell membranes in the manner of FIG. 5, but infact when cholestosomes are absorbed into enterocytes and then passedintact into chylomicrons, the delivery inside cells is much greater.These examples illustrating greater intracellular penetration will alsobe shown.

Specific Commercial Opportunities for Individual Molecules—

It is well known that prior disclosed delivery methods, and compositionsinvolving lipids in formulation do not pass the enterocytes of thegastrointestinal tract, are not incorporated into chylomicrons, and donot pass the membrane of most cells.

The advanced capabilities conferred by cholestosome encapsulation aresurprising in view of the composite prior art. Put simply, no deliverysystem has functioned favorably at the task of oral delivery of largehydrophilic molecules (especially macromolecules such as polypeptides(especially including monoclonal and polyclonal antibodies), proteinsand polynucleotides, especially RNA, including small interfering RNA,small hairpin RNA, microRNA, and DNA, especially including plasmid DNAand naked DNA.

Among these molecules, there are preferred candidates by virtue of theneed for chronic use in disease treatment simply because now they mustbe injected and subsequently after formulation into cholestosomes theymay be given by mouth, or applied to skin as an ointment or cream, orinhaled as a lipid nanoparticle. These will be disclosed.

Two Step Cholestosome Facilitated Delivery of Macromolecules into Cells.

In the Instant invention, as illustrated in FIG. 1, a macromolecule suchas insulin may be delivered inside body cells after two sequential stepsin formulation and bioprocessing. In the first step, we prepare acholestosome and encapsulate said macromolecule in the formedcholestosome, in full recognition that the cholestosome must becomprised of specific cholesteryl esters selected for compatibility witheach chosen molecule. The cholesteryl ester constituents of thecholestosome must also be selected to ensure that there is a transporteron the surface of duodenal enterocytes. The second step in preparationof the macromolecular delivery system occurs when the duodenalenterocytes incorporate the cholestosome-macromolecule construct intochylomicrons and secrete these now loaded and newly transformedchylomicrons into lymphatic fluids which carry the chylomicrons to thethoracic duct and eventually circulate in blood of said patient.

Docking Because of APO-B Incorporation into Chylomicrons

Intracellular delivery of macromolecules encapsulated withincholestosomes and incorporated within chylomicrons is accomplished whenthe chylomicrons containing the cargo-loaded cholestosome containing anactive molecule payload dock with cells in need of cholesterol andtriglycerides and transfer said components including said cholestosomesinto cells without requiring endosome encapsulation. A further noveltyof the present method is release of cholestosome incorporatedmacromolecules into cytoplasm of cells in intact form as exteriorsurface cholesteryl esters are removed in cytoplasm and thereby releasethe molecule from its cholestosome delivery capsule. As long as thesurface of the cholestosome is then recognized as a needed cholesterylester by the target cell, the target cell will receive the payload andafter unpacking the surface by cholesteryl ester hydrolases themacromolecule will be freed inside the membrane of the target body cell.

As can be appreciated from the images of MCF-7 cells in FIG. 22, thereis nearly always some uptake of molecules from cholestosomes alone, asshown with cholestosome encapsulated FITC (the control) in FIG. 5.However, there is an unexpectedly large (at least 1000× greater)differential uptake by MCF-7 cells when FITC cholestosomes are firstexposed to Caco-2 cells and the resulting FITC-cholestosome-chylomicronsare collected from the basolateral side of the apparatus (as in FIG.17), then used for exposure to MCF-7 cells. It should be noted thatthese FITC-insulin-cholestosome chylomicrons were prepared using theFITC-insulin-cholestosomes in the same amount on the Caco-2 cells, soessentially all the FITC-insulin-cholestosomes were incorporated by theCaco-2 cells into FITC insulin chylomicrons.

Loading of Cholestosomes into Chylomicrons

Loading of cholestosomes and their molecular payload into chylomicronsby the golgi apparatus appears to be quantitative, as evidenced byre-measurement of the apical side of the Caco-2 and subtraction of theamount remaining from the amount recovered in chylomicrons on thebasolateral side. Thus, the affinity of Caco-2 cells for cholestosomesappears to be very high. The Caco-2 cells clear all of the availablecholestosomes placed on the apical side into chylomicrons on thebasolateral side. Thus, the early choice of cholesteryl esters to beused to encapsulate the active molecule(s) is an essential step in thepractice of the invention.

Protection of Molecular Payloads from Acid and/or Enzymatic Degradationin the GI Tract.

Cholestosomes survive intact at pH values ranging from 2-14, in contrastto liposomes which are rapidly degraded by these same conditions and arerelatively unstable compared to compositions according to the presentinvention. Cholestosomes prepared with labile payloads may be coatedwith an outer enterically targeted layer in cases where their payloadconstituents must be protected from degradation in the gastrointestinaltract so that the cargo-loaded cholestosomes reach the duodenum (G.I.sites of enterocytes which produce chylomicrons incorporating thecholestosomes).

Payloads such as insulin and other proteins/polypeptides are acidlabile, necessitating an additional step of an enteric coatingprotective of insulin in cholestosomes, to be applied prior to use inanimal or in vivo systems where there is potential for acid or enzymaticdegradation. Under usual situations in the practice of the art, when thecontents of a cholestosome are acid labile peptides and proteins, andwhen these products are cholestosome encapsulated in preparation fororal ingestion, there should be a final product administered with anenteric coating to protect the contents of the cholestosomes from theacid in the stomach. In most cases after release of the cholestosomes inthe duodenum there is the possibility of enzymatic degradation or bilesalt mediated saponification in the duodenum, so there is a need toperform stability studies of the individual cholestosomes in contactwith bile salts, pancreatic lipases and pancreatic esterases. Thereforeunless or until the protein or peptide is definitively proven to be freeof acid degradation, the dosage form will be a small capsule filled withcholestosome construct, then coated with enteric coating to releasecontents at pH 5.5 to 6.0. A suitable coating for this purpose would beEudragit (64, 65) or another enteric polymer which is stable to acid buthaving similar degradation characteristics to the Eudragi polymers andwhile cholestosomes themselves are stable in low pH, there remains aneed to employ enteric coatings known in the art to protect the contentsof cholestosomes from acid degradation.

In certain embodiments, the pharmaceutical composition is a unilamellarvesicle having a diameter of about 100 to about 750 nm, preferably about225 to about 275 nm, and even more preferably around 250 nm. DLLSmeasurements indicate vesicles with diameters ranging from 50 nm to morethan 1000 nm. The final size used can be made from selective extrusionwith an appropriate pore size as well as control of time of sonicationas well as other preparation parameters.

“Immunomicelles” and “micelles” are aggregates formed by amphipathicmolecules in water or an aqueous solvent such that their polar ends orportions are in contact with the water or aqueous solvent and theirnonpolar ends or portions are in the interior of the aggregate. Amicelle can take any shape or form, including but not limited to, anon-lamellar “detergent-like” aggregate that does not enclose a portionof the water or aqueous solvent, or a unilamellar or multilamellar“vesicle-like” aggregate that encloses a portion of the water or aqueoussolvent, such as, for example, a liposome. Specifically included withinthe definition of “micelle” are small unilamellar vesicles or liposomes(“SUVs”), small multilamellar vesicles or liposomes (“SMVs”), largeunilamellar vesicles or liposomes (“LUVs”) and large multilamellarvesicles or liposomes (“LMVs”).

As defined in United States Patent Application Document No. 20110268653“‘lipidic particle’ refers to a particle having a membrane structure inwhich amphipathic lipid molecules are arranged with their polar groupsoriented to an aqueous phase. Examples of the lipid membrane structureinclude configurations such as a liposome, multi-lamellar vesicle (MLV),and a micelle structure. A ‘liposome’ refers to a closed nanosphere,which is formed by forming a bilayer membrane of a phospholipid moleculewith the hydrophobic moiety positioned inside and the hydrophilic moietypositioned outside, in water and closing the ends of the bilayermembrane. Examples of liposomes include a nanosphere having a singlelayer formed of a phospholipid bilayer membrane and a nanosphere havinga multiple layer formed of a plurality of phospholipid bilayers. Since aliposome has such a structure, an aqueous solution is present bothinside and outside of the liposome and the lipid bilayer serves as theboundary. A ‘micelle’ refers to an aggregate of amphipathic molecules.The micelle has a form in which a lipophilic moiety of this amphipathicmolecules is positioned toward the center of the micelle and ahydrophilic moiety is positioned toward the outside thereof, in anaqueous medium. A center of a sphere is lipophilic and a peripheralportion is hydrophilic in such a micelle. Examples of a micellestructure include spherical, laminar, columnar, ellipsoidal, microsomaland lamellar structures, and a liquid crystal.” Note that suchstructures do a very poor job of encapsulating hydrophilic moleculeslike peptides and proteins, where loading is 1:1000 or worse. Contrastthat with cholestosomes with hydrophilic centers (from the orientationof the ester functionality) and hydrophobic outsides. In certainembodiments, the interior and exterior may be the same with the sterolnucleus on the outside surface and inside cavity with the tails of theesters interdigitated in a Pseudo-bilayer type of molecule. When achylomicron takes up a cholestosome, the truly hydrophilic outside isre-established by the Apolipoprotein components of the transformed andloaded chylomicrons, and the Apolipoproteins also facilitate docking ofthe transformed chylomicrons with cells. In short, the cholestosome twostage formation into a chylomicron is totally novel and unexpectedcompared to previous efforts.

The term “patient” or “subject” is used throughout the specification todescribe an animal, preferably a human, to whom treatment, includingprophylactic treatment, with the compositions according to the presentinvention is provided. For treatment of those infections, conditions ordisease states which are specific for a specific animal such as a humanpatient, the term patient refers to that specific animal.

The term “compound” is used herein to refer to any specific chemicalcompound or disclosed herein. Within its use in context, the termgenerally refers to a single small molecule as disclosed herein, but incertain instances may also refer to stereoisomers and/or optical isomers(including racemic mixtures) of disclosed compounds. The term compoundincludes active metabolites of compounds and/or pharmaceutically activesalts thereof.

The term “active molecule”, “active agent” or “active compound” shallmean any molecule which is active in a biological system and which maybe incorporated into a cholestosome as described herein. Cholestsomesaccording to the present invention are able to readily accommodate alarge number of active compounds, including small molecules and largemolecules, especially including compounds which cannot otherwise bedelivered efficiently orally. This is because of the unique mechanism(as described herein) that cargo-loaded cholestosomes provide indelivering active compounds through enterocytes into chylomicrons andthen into the cells of a patient or subject to whom these cargo-loadedcholestosomes are administered. These active molecules include smallmolecules which are unstable to standard oral delivery techniques andare typically only parenterally administered and macromolecules such asproteins (including glycoproteins) and polypeptides (e.g insulin,interferon, hCG, C-reactive protein, cytokines, including variousinterleukins, growth factors), other polypeptides, including antibodiessuch as polyclonal antibodies, monoclonal antibodies (as otherwisedescribed in detail herein, antibody fragments (single chain variablefragments or scFv, antigen-binding fragments or Fab, ₃G antibodies),immunogenic polypeptides and oligopeptides, polynucleotides, includingDNA and RNA, such as naked DNA, plasma DNA, mRNA, siRNA, shRNA,bifunctional shRNA, microRNA (including miR-122, among others) andvarious oligonucleotides of DNA and RNA. Numerous anti-infective agents,including antibiotics (such as vancomycin and penicillin) and antiviralagents and other active molecules, especially including macromolecularantibiotics as well as numerous anticancer agents which are disclosed indetail herein, may also be delivered by the present invention. It isnoted that cholestosomes pursuant to the present invention may be usedto deliver virtually any active molecule of a wide variety of sizes andmolecular weight. Cholestosomes according to the present invention mayalso be used to topically deliver a number of active molecules toprovide high bioavailability through the skin of a patient or subjectincluding topical antibiotics, topical anti-fungals, topical plateletderived growth factor, other growth factors, topical anti-TNF forpsoriasis, for example and topical vaccines, and topical deliver ofcosmetic agents, among others. Numerous chemotherapeutic agents,antibiotics, and antiviral agents may be incorporated into cholestosomesaccording to the present invention. The cholestosomes according to thepresent invention are particularly suited for these compounds, evensmall molecules, because delivery of the compound into the cell pursuantto the mechanism of active molecule delivery by compositions accordingto the present invention represents a particularly effective therapyagainst a variety of microbes, including bacteria and viruses.

Antifungals for use in the present invention include, for example,miconazole, terconazole, econazole, isoconazole, tioconazole,bifonazole, clotrimazole, ketoconazole, butaconazole, itraconazole,oxiconazole, fenticonazole, nystain, naftifine, amphotericin B,zinoconazole, ciclopiroxolamine or a mixture thereof

Antibiotics for use in the present invention include Aminoglycosides,including Gentamicin Garamycin Kanamycin, Neomycin, Netilmicin,Tobramycin, Paromomycin, Spectinomycin; Ansamycins, includingGeldanamycin, Herbimycin Rifaximin and Streptomycin; Carbapenems,including Ertapenem Doripenem Imipenem/Cilastatin and Meropenem;Cephalosporins, including Cefadroxil Cefazolin Cefalotin/Cefalothin,Cefalexin Cefactor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime,Cefixime Cefdinir, Cefditoren, Cefoperazone Cefotaxime Cefpodoxime,Ceftazadime, Ceftibuten Ceftizoxime Ceftriaxone, Cefipime, Ceftarolinefosamil and Ceftobiprole; Glycopeptides, including Teicoplanin,Vancomycin and Telavancin; Lincosamides, including Clindamycin andLincomycin; Lipopeptipdes, including Daptomycin, Oritavancin, WAP-8294A;Macrolides, including Azithromycin, Clarithromycin, Dirithromycin,Erythromycin, Roxithromycin, Telithromycin and Spiramycin;

Monobactams, including Aztreonam; Nitrofurans, including Furazolidoneand Nitrofurantoin; Oxazolidonones, including Linezolid, Posizolid,Radezolid and Torezolid;Penicillins, including Amoxicillin, Ampicillin, Azlocillin,Carbenacillin, Cloxacillin Dicloxacillin, Flucloxacillin, Mezlocillin,Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V,Piperacillin, Temocillin and Ticarcillin; Penicillin combinationsincluding Amoxicillin/clavulanate, Ampicillin/sulbactam,Piperacillin/tazobactam and Ticarcillin/clavulanate; Polypeptides,including Bacitracin, Colistin and Polymyxin B;Quinolones/fluoroquinolines, including Ciprofloxacin, Enoxacin,Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin,Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin,Sparfloxacin and Temafloxacin;Sulfonamides, including Mafenide, Sulfacetamide, Sulfadiazine,Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfasalazine,Sulfisoxazole, Trimethoprim-Sulfamethoxazole and Sulfonamidochrysoidine;Tetracyclines, including Demeclocycline, Doxycycline, VibramycinMinocycline, Oxytetracycline and Tetracycline; Anti-mycobacterial,including Clofazimine, Capreomycin, Cycloserine, Ethambutol, Rifampicin,Rifabutin, Rifapentine, Arsphenamine, Chloramphenicol, Fosfomycin,Fusidic acid, Metronidazole, Mupirocin, Platensimycin,Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline, Tinidazole andTrimethoprim.

Antiviral agents include anti-HIV agents, anti-HBV agents and anti-HCVagents which are known in the art. Anti-HIV agents include 3TC(Lamivudine), AZT (Zidovudine), (−)-FTC, ddI (Didanosine), ddC(zalcitabine), abacavir (ABC), tenofovir (PMPA), D-D4FC (Reverset), D4T(Stavudine), Racivir, L-FddC, L-FD4C (Elvucitabine), Festinavir, NVP(Nevirapine), DLV (Delavirdine), EFV (Efavirenz), SQVM (Saquinavirmesylate), RTV (Ritonavir), IDV (Indinavir), SQV (Saquinavir), NFV(Nelfinavir), APV (Amprenavir), LPV (Lopinavir), fusion inhibitors suchas T20, among others, fuseon and mixtures thereof, including anti-HIVcompounds presently in clinical trials or in development.

Other anti-HIV agents which may be used in the present inventioninclude, various NNRTI's selected from the group consisting ofnevirapine (BI-R6-587), delavirdine (U-90152S/T), efavirenz (DMP-266),UC-781(N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2methyl3-furancarbothiamide),etravirine (TMC125), Trovirdine (Ly300046.HCl), MKC-442 (emivirine,coactinon), HI-236, H-240, H-280, HI-281, rilpivirine (TMC-278),MSC-127, HBY 097, DMP266, Baicalin (TJN-151), U-104489 or PNU-104489),Capravirine, atevirdine Calanolide A (NSC675451), Calanolide B andFoscarnet (Foscavir), among others.

Anti-HBV agents which may be formulated in cholestosomes according tothe present invention include Hepsera (adefovir dipivoxil), lamivudine,entecavir, teibivudine, tenofovir, emtricitabine, clevudine,valtoricitabine, arndoxovir, pradefovir, racivir, BAM 205, nitazoxanide,UT 231-B, Bay 41-4109, EHT899, zadaxin (thymosin alpha-1) and mixturesthereof.

Anti-HCV agents which may be formulated in cholestosomes according tothe present invention include ribavirin, interferon, pegylatedinterferon, boceprevir, daclatasvir, asunapavir, INX-189, FV-100, NM283, VX-950 (telaprevir), SCH 50304, TMC435, VX-500, BX-813, SCH503034,R1626, ITMN-191 (R7227), R7128, PF-868554, TT033, CGH-759, GI 5005,MK-7009, SIRNA-034, MK-0608, A-837093, GS 9190, GS 9256, GS 9451, GS5885, GS 6620, GS 9620, GS9669, ACH-1095, ACH-2928, GSK625433, TG4040(MVA-HCV), A-831, F351, NS5A, NS4B, ANA598, A-689, GNI-104, IDX102,ADX184, ALS-2200, ALS-2158, BI 201335, BI 207127, BIT-225, BIT-8020,GL59728, GL60667, PSI-938, PSI-7977, PSI-7851, SCY-635, TLR9 Agonist,PHX1766, SP-30 and mixtures thereof.

Other compounds for use in the present invention are also describedherein below, in the examples which follow.

It is noted in the present invention that incorporation of activemolecules into cholestosomes and administration to a patient or subjectwill produce a greater therapeutic effect at the same dosage level thanidentical active molecules delivered by prior art methods. In effect,the mechanism of packaging cargo-loaded cholestosomes in chylomicronsresults in a substantial greater amount or concentration of an activemolecule at its actual site of activity (in a cell) resulting insubstantially greater efficacy than prior art methods. In manyinstances, the amount of concentration of active agent delivered insidea cell according to the present invention is at least 2 and often asmuch as 10 times to 1000 times the concentration of active compared todelivery by prior art (contemporary) means.

The term “effective amount” is used throughout the specification todescribe concentrations or amounts of formulations or other componentswhich are used in amounts, within the context of their use, to producean intended effect according to the present invention. The formulationsor component may be used to produce a favorable change in a disease orcondition treated, whether that change is a remission, a favorablephysiological result, a reversal or attenuation of a disease state orcondition treated, the prevention or the reduction in the likelihood ofa condition or disease-state occurring, depending upon the disease orcondition treated. Where formulations are used in combination, each ofthe formulations is used in an effective amount, wherein an effectiveamount may include a synergistic amount. The amount of formulation usedin the present invention may vary according to the nature of theformulation, the age and weight of the patient and numerous otherfactors which may influence the bioavailability and pharmacokinetics ofthe formulation, the amount of formulation which is administered to apatient generally ranges from about 0.001 mg/kg to about 50 mg/kg ormore, about 0.5 mg/kg to about 25 mg/kg, about 0.1 to about 15 mg/kg,about 1 mg to about 10 mg/kg per day and otherwise described herein. Foravoidance of doubt, the dosage of the component in said formulationgiven to said animal is approximately the same as would be given byparenteral means, after correction for the added mass of the deliverysystem. The person of ordinary skill may easily recognize variations indosage schedules or amounts to be made during the course of therapy.

The term “coadministration” is used to describe the administration oftwo or more active compounds, in this case a compound according to thepresent invention, in combination with an additional agent or otherbiologically active agent, in effective amounts. Although the termcoadministration preferably includes the administration of two or moreactive compounds to the patient at the same time, it is not necessarythat the compounds actually be administered at the exact same time or inthe same composition (although that may be preferable), only thatamounts of compound will be administered to a patient or subject suchthat effective concentrations are found in the blood, serum or plasma,or in the pulmonary tissue at the same time.

The term “Ileal brake hormone releasing agent” is used to describe anutritional substance which modules hormones in the ileum. Thesenutritional substances include, but are not limited to proteins andassociated amino acids, fats including saturated fats, monosaturatedfats, polyunsaturated fats, essential fatty acids, Omega-3 and Omega-6fatty acids, trans fatty acids, cholesterol, fat substitutes,carbohydrates such as dietary fiber (both soluble and insoluble fiber),starch, sugars (including monosaccharidesmonosaccharide, fructose,galactose, glucose, disacharidesdisaccharides, lactose, maltose,sucrose, and alcohol), polymeric sugars including inulin andpolydextrose, natural sugar substitutes (including brazzein. Curculin,erythritol, fructose, glycyrrhizin, glycyrrhizin, glycerol, hydrogenatedstarch hydrosylates, isomalt, lactitol, mabinlin, maltitol, mannitol,miraculin, monellin, pentadin, sorbitol, stevia, tagatose, thaumatin,and xylitol), sahlep, and halwa root extract. D-glucose (dextrose) is apreferred nutritional substance. Nutritional substances include allcompositions that yield the aforementioned nutrients upon digestion orthat contain such nutrients, including polymeric forms of thesenutrients. When these compositions are related in the ileum of apatient, ileal brake hormones are modulated often resulting in anincrease in activity, thus providing a beneficial therapeutic effect ona number of disease states and conditions. The effects of ileal brakehormone releasing agents are discussed in great detail in US patentpublication 2011-0268795, published Nov. 3, 2011 and internationalpatent publications WO 2010/027498, published Mar. 10, 2010 and WO2013/063527, published May 2, 2013 and may be referenced for theteachings which are incorporated therein. A preferred ileal brakehormone releasing agent is glucose administered in the ileum of apatient at a dose ranging from about 7.5 g to about 12 g or more.

The term “ileum hormones” includes all hormones that are associated withintraluminal food substances stimulating the release of said hormones,which may be caused by ileum-related stimulation of insulin secretion orinhibition of glucagon secretion or the delivery of a nutritionalsubstance within the teachings of the art to the ileum of a patient orsubject. “Ileum hormones” therefore include, but are not limited to,GLP-1, glicentin, C-terminally glycine-extended GLP-1 (7 37), (PG (78108)); intervening peptide-2 (PG (111 122) amide); GLP-2 (PG (126 158),GRPP (PG (1 30)), oxyntomodulin (PG (33 69), and other peptide fractionsto be isolated, PYY (PYY 1-36) and (PYY 3-36), cholecystokinin (CCK),gastrin, enteroglucagon and secretin.

The term “ileum hormone-stimulating amount of a nutritional substance”means any amount of a nutritional substance that is effective to inducemeasurable hormone release in the ileum, and induce satiety feedbackfrom the ileum or ileum-related stimulation of insulin secretion orinhibition of glucagon secretion, or other effect such as shutting downor decreasing insulin. resistance and increasing glucose tolerance.Consequently, an “ileum hormone-stimulating amount of a nutritionalsubstance” can vary widely in dosage depending upon factors such as thespecific nutrient at issue, the desired effect of administration, thedesired goal of minimizing caloric intake, and the characteristics ofthe subject to whom the nutritional substance is administered. Forexample, at least about 500 mg of D-glucose is used, and a particularlypreferred ileum hormonal-stimulating amount of D-glucose includesbetween about 7.5-8 g to about 12-12.5 g or more (preferably around 10g).

Additional nutritional components which may be included in compositionsaccording to the present invention include, barley grass, known to be arich source of highly metabolizable vitamins and minerals such asvitamins A, B1, B2, B6, and C, potassium, magnesium, and zinc. Inaddition, barley grass also has a high concentration of the enzymesuperoxide dismutase (SOD), which has been shown to have high levels ofantioxidant activity. Barley grass is believed to be an importantnutrient in the regulation of the digestive process because themicronutrients, enzymes (e.g., SOD), and fiber contained in barley grassare believed to improve intestinal function.

Alfalfa fresh or dried leaf tea is also usable in the invention, topromote appetite, and as a good source of chlorophyll and fiber. Alfalfacontains biotin, calcium, choline, inositol, iron, magnesium, PABA,phosphorus, potassium, protein, sodium, sulfur, tryptophan (amino acid),and vitamins A, B complex, C, D, E, K, P, and U. Alfalfa supplements arerecommended for treating poor digestion, and were shown to lowercholesterol levels in animal studies. Alfalfa is categorized asGenerally Regarded as Safe (GRAS) by the FDA. Dosages can range from25-1500 mg, preferably 500-1000 mg dried leaf per day.

Chlorella is yet another substance usable in the invention incombination with the nutritional substance (preferably D-glucose ordextrose), being a genus of unicellular green algae, grown and harvestedin tanks, purified, processed and dried to form a powder. Chlorella isrich in chlorophyll, carotenes, and contains the full vitamin B complex,vitamins E and C, and has a wide range of minerals, including magnesium,potassium, iron and calcium. Chlorella also provides dietary fiber,nucleic acids, amino acids, enzymes, CGF (Chlorella Growth Factor) andother substances. Dosages can range from 300-1500 mg/day.

Chlorophyllin is yet another nutritional substance, being a known foodadditive and has been used as an alternative medicine. Chlorophyllin isa water-soluble, semi-synthetic sodium/copper derivative of chlorophyll,and the active ingredient in a number of internally-taken preparationsintended to reduce odors associated with incontinence, colostomies andsimilar procedures, as well as body odor in general. It is alsoavailable as a topical preparation, purportedly useful for treatment andodor control of wounds, injuries, and other skin conditions, such as forradiation burns.

Sodium alginate may also be used as a nutritional substance, preferablyin combination with D-glucose or dextrose.

The term “prophylactic” is used to describe the use of a formulationdescribed herein which reduces the likelihood of an occurrence of acondition or disease state in a patient or subject. The term “reducingthe likelihood” refers to the fact that in a given population ofpatients, the present invention may be used to reduce the likelihood ofan occurrence, recurrence or metastasis of disease in one or morepatients within that population of all patients, rather than prevent, inall patients, the occurrence, recurrence or metastasis of a diseasestate.

The term “pharmaceutically acceptable” refers to a salt form or otherderivative (such as an active metabolite or pro-drug form) of thepresent compounds or a carrier, additive or excipient which is notunacceptably toxic to the subject to which it is administered.

Inflammation Associated Metabolic Disorder

An “inflammation-associated metabolic disorder” includes, but is notlimited to, lung diseases, hyperglycemic disorders including diabetesand disorders resulting from insulin resistance, such as Type I and TypeII diabetes, as well as severe insulin resistance, hyperinsulinemia, anddyslipidemia (e.g. hyperlipidemia (e.g., as expressed by obesesubjects), elevated low-density lipoprotein (LDL), depressedhigh-density lipoprotein (HDL), and elevated triglycerides) and Hepatic(or liver) injury associated with insulin resistance and inflammationincluding Hepatic steatosis, Non alcoholic fatty liver diseases,cirrhosis, hepatitis caused by viruses such as Hepatitis A, B, or C, ortoxins and insulin-resistant diabetes, such as Mendenhall's Syndrome,Werner Syndrome, leprechaunism, and lipoatrophic diabetes, renaldisorders, such as acute and chronic renal insufficiency, end-stagechronic renal failure, glomerulonephritis, interstitial nephritis,pyelonephritis, glomerulosclerosis, e.g., Kimmelstiel-Wilson in diabeticpatients and kidney failure after kidney transplantation, obesity,GH-deficiency, GH resistance, Turner's syndrome, Laron's syndrome, shortstature, increased fat mass-to-lean ratios, immunodeficiencies includingdecreased CD4⁺ T cell counts and decreased immune tolerance orchemotherapy-induced tissue damage, bone marrow transplantation,diseases or insufficiencies of cardiac structure or function such asheart dysfunctions and congestive heart failure, atherosclerosis,neuronal, neurological, or neuromuscular disorders, e.g., diseases ofthe central nervous system including Alzheimer's disease, or Parkinson'sdisease or multiple sclerosis, and diseases of the peripheral nervoussystem and musculature including peripheral neuropathy, musculardystrophy, or myotonic dystrophy, and catabolic states, including thoseassociated with wasting caused by any condition, including, e.g., mentalhealth condition (e.g., anorexia nervosa), trauma or wounding orinfection such as with a bacterium or human virus such as HIV, HepatitisC or B, wounds, skin disorders, gut structure and function that needrestoration, and so forth.

An “inflammation-associated metabolic disorder” also includes a cancerand an “infectious disease” as defined herein, as well as disorders ofbone or cartilage growth in children, including short stature, and inchildren and adults disorders of cartilage and bone in children andadults, including arthritis and osteoporosis. An“inflammation-associated metabolic disorder” includes a combination oftwo or more of the above disorders (e.g., osteoporosis that is asequelae of a catabolic state). Specific disorders of particularinterest targeted for treatment herein are diabetes and obesity, heartdysfunctions, kidney disorders, neurological disorders, bone disorders,whole body growth disorders, and immunological disorders.

In one embodiment, “inflammation-associated metabolic disorder”includes: central obesity, dyslipidemia including particularlyhypertriglyceridemia, low HDL cholesterol, small dense LDL particles andpostprandial lipemia; glucose intolerance such as impaired fastingglucose; insulin resistance and hypertension, and diabetes. The term“diabetes” is used to describe diabetes mellitus type I or type II. Thepresent invention relates to a method for improving pancreatic function,hepatic function, brain function, gastrointestinal function,cardiovascular function, renal function and symptoms, conditions anddisease states which occur secondary to impaired function in patients orsubjects with diabetes as otherwise described herein. It is noted thatin diabetes mellitus type I and II, renal function is impaired fromcollagen deposits, and not from cysts in the other disease statestreated by the present invention.

Mycobacterial infections often manifest as diseases such astuberculosis. Human infections caused by mycobacteria have beenwidespread since ancient times, and tuberculosis remains a leading causeof death today. Although the incidence of the disease declined, inparallel with advancing standards of living, since the mid-nineteenthcentury, mycobacterial diseases still constitute a leading cause ofmorbidity and mortality in countries with limited medical resources.Additionally, mycobacterial diseases can cause overwhelming,disseminated disease in immunocompromised patients. In spite of theefforts of numerous health organizations worldwide, the eradication ofmycobacterial diseases has never been achieved, nor is eradicationimminent. Nearly one third of the world's population is infected withmycobacterium tuberculosis complex, commonly referred to as tuberculosis(TB), with approximately 8 million new cases, and two to three milliondeaths attributable to TB yearly. Tuberculosis (TB) is the cause of thelargest number of human deaths attributable to a single etiologic agent(see Dye et al., J. Am. Med. Association, 282, 677-686, (1999); and 2000WHO/OMS Press Release).

Mycobacteria other than M. tuberculosis are increasingly found inopportunistic infections that plague the AIDS patient. Organisms fromthe M. avium-intracellulare complex (MAC), especially serotypes four andeight, account for 68% of the mycobacterial isolates from AIDS patients.Enormous numbers of MAC are found (up to 10¹⁰ acid-fast bacilli per gramof tissue), and consequently, the prognosis for the infected AIDSpatient is poor.

In many countries the only measure for TB control has been vaccinationwith M. Bovis bacille Calmette-Guerin (BCG). The overall vaccineefficacy of BCG against TB, however, is about 50% with extremevariations ranging from 0% to 80% between different field trials. Thewidespread emergence of multiple drug-resistant M. tuberculosis strainsis also a concern.

M. tuberculosis belongs to the group of intracellular bacteria thatreplicate within the phagosomal vacuoles of resting macrophages, thusprotection against TB depends on T cell-mediated immunity. Severalstudies in mice and humans, however, have shown that Mycobacteriastimulate antigen-specific, major histocompatibility complex (MHC) classII- or class I-restricted CD4 and CD8 T cells, respectively. Theimportant role of MHC class I-restricted CD8 T cells was convincinglydemonstrated by the failure of β2-microglobulin) deficient mice tocontrol experimental M. tuberculosis infection.

As used herein, the term “tuberculosis” comprises disease states usuallyassociated with infections caused by mycobacteria species comprising M.tuberculosis complex. The term “tuberculosis” is also associated withmycobacterial infections caused by mycobacteria other than M.tuberculosis. Other mycobacterial species include M.avium-intracellulare, M. kansarii, M. fortuitum, M. chelonae, M. leprae,M. africanum, and M. microti, M. avium paratuberculosis, M.intracellulare, M. scrofulaceum, M. xenopi, M. marinum, M. ulcerans.

Infectious Disease

An “infectious disease” includes but is limited to those caused bybacterial, mycological, parasitic, and viral agents. Examples of suchinfectious agents include the following: staphylococcus,streptococcaceae, neisseriaaceae, cocci, enterobacteriaceae,pseudomonadaceae, vibrionaceae, campylobacter, pasteurellaceae,bordetella, francisella, brucella, legionellaceae, bacteroidaceae,gram-negative bacilli, clostridium, corynebacterium, propionibacterium,gram-positive bacilli, anthrax, actinomyces, nocardia, mycobacterium,treponema, borrelia, leptospira, mycoplasma, ureaplasma, rickettsia,chlamydiae, systemic mycoses, opportunistic mycoses, protozoa,nematodes, trematodes, cestodes, adenoviruses, herpesviruses,poxviruses, papovaviruses, hepatitis viruses, orthomyxoviruses,paramyxoviruses, coronaviruses, picornaviruses, reoviruses, togaviruses,flaviviruses, bunyaviridae, rhabdoviruses, human immunodeficiency virusand retroviruses, among numerous others.

In certain embodiments, an “infectious disease” is selected from thegroup consisting of tuberculosis, leprosy, Crohn's Disease, acquiredimmunodeficiency syndrome, Lyme disease, cat-scratch disease, RockyMountain spotted fever and influenza.

Cancer

The term “cancer” is used throughout the specification to refer to thepathological process that results in the formation and growth of acancerous or malignant neoplasm, i.e., abnormal tissue that grows bycellular proliferation, often more rapidly than normal and continues togrow after the stimuli that initiated the new growth cease. Malignantneoplasms show partial or complete lack of structural organization andfunctional coordination with the normal tissue and most invadesurrounding tissues, metastasize to several sites, and are likel_(y) torecur after attempted removal and to cause the death of the patientunless adequately treated. As used herein, the term neoplasia is used todescribe all cancerous disease states and embraces or encompasses thepathological process associated with malignant hematogenous, ascetic andsolid tumors.

Components in Formulations of the Invention

Formulations of the invention may include a pharmaceutically acceptablediluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.Acceptable formulation materials preferably are nontoxic to recipientsat the dosages and concentrations employed. The pharmaceuticalformulations may contain materials for modifying, maintaining orpreserving, for example, the pH, osmolarity, viscosity, clarity, color,isotonicity, odor, sterility, stability, rate of dissolution or release,adsorption or penetration of the composition. Suitable formulationmaterials include, but are not limited to, amino acids (such as glycine,glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants(such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite);buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates orother organic acids); bulking agents (such as mannitol or glycine);chelating agents (such as ethylenediamine tetraacetic acid (EDTA));complexing agents (such as caffeine, polyvinylpyrrolidone,beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers;monosaccharides, disaccharides, and other carbohydrates (such asglucose, mannose or dextrins); proteins (such as serum albumin, gelatinor immunoglobulins); coloring, flavoring and diluting agents;emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone);low molecular weight polypeptides; salt-forming counterions (such assodium); preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide);solvents (such as glycerin, propylene glycol or polyethylene glycol);sugar alcohols (such as mannitol or sorbitol); suspending agents;surfactants or wetting agents (such as pluronics, polyethylene glycol(PEG), sorbitan esters, polysorbates such as polysorbate 20 andpolysorbate 80, Tween was used in the present example; Tween may be usedto improve the yield of emulsion prior to extrusion step; Tween can beadded to the aqueous preparation prior to the addition to the lipids orto the lipid and then addition of aqueous. The smallest amount of tweenpossible is used, that being less than about 100 microliters in 10 ml ofaqueous. Triton, trimethamine, lecithin, cholesterol, or tyloxapal);stability enhancing agents (such as sucrose or sorbitol); tonicityenhancing agents (such as alkali metal halides, preferably sodium orpotassium chloride, mannitol, or sorbitol); delivery vehicles; diluents;excipients and/or pharmaceutical adjuvants. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, 18.sup.th Edition, (A. R. Gennaro,ed.), 1990, Mack Publishing Company.

Optimal pharmaceutical formulations can be determined by one skilled inthe art depending upon, for example, the intended route ofadministration, delivery format and desired dosage. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, Id. Such formulations may influencethe physical state, stability, rate of in vivo release and rate of invivo clearance of the antibodies of the invention.

Primary vehicles or carriers in a pharmaceutical formulation caninclude, but are not limited to, water for injection, physiologicalsaline solution or artificial cerebrospinal fluid, possibly supplementedwith other materials common in compositions for parenteraladministration. Neutral buffered saline or saline mixed with serumalbumin are further exemplary vehicles. Pharmaceutical formulations cancomprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH4.0-5.5, which may further include sorbitol or a suitable substitute.Pharmaceutical formulations of the invention may be prepared for storageby mixing the selected composition having the desired degree of puritywith optional formulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES,Id.) in the form of a lyophilized cake or an aqueous solution. Further,the formulations may be formulated as a lyophilizate using appropriateexcipients such as sucrose.

Formulation components are present in concentrations that are acceptableto the site of administration. Buffers are advantageously used tomaintain the composition at physiological pH or at a slightly lower pH,typically within a pH range of from about 5 to about 8.

The pharmaceutical formulations of the invention can be deliveredparenterally. When parenteral administration is contemplated, thetherapeutic formulations for use in this invention may be in the form ofa pyrogen-free, parenterally acceptable aqueous solution. Preparationinvolves the formulation of the desired immunomicelle, which may providecontrolled or sustained release of the product which may then bedelivered via a depot injection. Formulation with hyaluronic acid hasthe effect of promoting sustained duration in the circulation.

Formulations according to the present invention may be formulated forinhalation. In these embodiments, a stealth Cholestosome-moleculeformulation is formulated as a dry powder for inhalation, or inhalationsolutions may also be formulated with a propellant for aerosol delivery,such as by nebulization. Pulmonary administration is further describedin PCT Application No. PCT/US94/001875, which describes the pulmonarydelivery of chemically modified proteins and is hereby incorporated byreference.

Formulations may be formulated for topical application on the skin. Inthese embodiments, a stealth Cholestosome-molecule formulation isformulated as an ointment or cream, and applied to the surface of theskin.

Formulations of the invention can be delivered through the digestivetract, such as orally and this represents a preferred route ofadministration. The preparation of such pharmaceutically acceptablecompositions is disclosed herein and within the skill of the art.Formulations disclosed herein that are administered in this fashion maybe formulated with or without those carriers customarily used in thecompounding of solid dosage forms such as tablets and capsules. Acapsule may be designed to release the active portion of the formulationat the point in the gastrointestinal tract when bioavailability ismaximized and pre-systemic degradation is minimized. Enteric coatingswhich are stable to acid but degradable within a pH of the duodenum(about 5.0 to about 6.0 or slightly higher) may be preferred. These arewell known in the art. Additional agents can be included to facilitateabsorption. Diluents, flavorings, low melting point waxes, vegetableoils, lubricants, suspending agents, tablet disintegrating agents, andbinders may also be employed.

A formulation may involve an effective quantity of a cholestosome, mostpreferentially a cholestosome formulation and a molecule in apharmaceutical composition as disclosed herein in a mixture withnon-toxic excipients that are suitable for the manufacture of tablets.By dissolving the tablets in sterile water, or another appropriatevehicle, solutions may be prepared in unit-dose form. Suitableexcipients include, but are not limited to, inert diluents, such ascalcium carbonate, sodium carbonate or bicarbonate, lactose, or calciumphosphate; or binding agents, such as starch, gelatin, or acacia; orlubricating agents such as magnesium stearate, stearic acid, or talc.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this may be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method may be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration may be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierce-able by ahypodermic injection needle.

Once the formulation of the invention has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations may bestored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration.

Administration routes for formulations of the invention include orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,intra-ocular, intraarterial, intraportal, or intralesional routes; bysustained release systems or by implantation devices. The pharmaceuticalformulations may be administered by bolus injection or continuously byinfusion, or by implantation device. The pharmaceutical formulationsalso can be administered locally via implantation of a membrane, spongeor another appropriate material onto which the desired molecule has beenabsorbed or encapsulated. Where an implantation device is used, thedevice may be implanted or topically applied into any suitable tissue ororgan, and delivery of the desired molecule may be via diffusion,timed-release bolus, or continuous administration.

As used herein, “enteric coatings” are substantially insoluble at a pHof less than a range of between about 5.0 to 7.0 to about 7.6(preferably about 5.0 to about 6.0 or slightly more within this ragne),and can be comprised of a variety of materials, including but notlimited to one or more compositions selected from the group consistingof poly(dl-lactide-co-glycolide, chitosan (Chi) stabilized with PVA(poly-vinylic alcohol), a lipid, an alginate,carboxymethylethylcellulose (CMEC), cellulose acetate trimellitiate(CAT), hydroxypropylmethyl cellulose phthalate (HPMCP),hydroxypropylmethyl cellulose, ethyl cellulose, color con, food glazeand mixtures of hydroxypropylmethyl cellulose and ethyl cellulose,polyvinyl acetate phthalate (PVAP), cellulose acetate phthalate (CAP),shellac, copolymers of methacrylic acid and ethyl acrylate, andcopolymers of methacrylic acid and ethyl acrylate to which a monomer ofmethylacrylate has been added during polymerization.

Enteric coatings can be applied by conventional coating techniques, suchas pan coating or fluid bed coating, using solutions of polymers inwater or suitable organic solvents or by using aqueous polymerdispersions. As an alternative embodiment, the release controllingenteric coating can separate additional antigen and/or drug layers onthe core; for instance, after coating with the release controllingsubstance, another antigen and/or drug layer can be applied, which isfollowed by another release controlling layer, etc. For example,suitable materials for the release controlling layer include EUDRAGIT®(copolymers of acrylic and methacrylic acid esters), EUDRAGIT®RS(copolymers of acrylic and methacrylic acid esters), cellulosederivatives such as ethylcellulose aqueous dispersions (AQUACOAT®,SURELEASE®), hydroxyethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, polyvinylpyrrolidone,polyvinylpyrrolidone/vinyl acetate copolymer, OPADRY®, and the like.

These and other aspects of the invention are described further in thefollowing non-limiting examples.

NON-LIMITING EXAMPLES OF THE PRESENT APPLICATION Example 1 Step by Step:General Preparation and Testing of Cholestosome Encapsulated Proteins,Peptides and Genetic Material Cholestosomes Applied to an Oral Proteinor Peptide

Steps in the preparation of an oral drug molecule, oral protein, oralpeptide, oral gene or construct of genetic material (the term “molecule”used to define one or all of these hereinafter in this example) andtesting of said molecule for absorption in Caco2 cells are as follows:

-   -   1. Prepare cholesteryl esters and composition elements for        encapsulation;    -   2. Obtain molecule targeted for encapsulation and test for        purity and stability at 37 C-45° C.;    -   3. Optimize components of cholesteryl esters in the cholestosome        mixture using a computer model of interactions between esters        and molecule to achieve maximum cholestosome loading of said        molecule;    -   4. Prepare cholestosome encapsulated molecule and include        Fluorescein Isothicyanate (FITC) label for purposes of        conducting biological studies including microscopy, said FITC        label not a component of product intended for human testing or        therapeutic use;    -   5. Test FITC labeled molecule in Caco2 cell monolayer and        collect chylomicron encapsulated FITC-cholestosome-molecules,        now defined as incorporated into cholestosome loaded        chylomicrons;    -   6. Expose test cells to chylomicrons containing        FITC-cholestosome-molecules and determine uptake of        FITC-molecule by these test cells. While MCF-7 cells are often        chosen because of their ease of use and relevance to cancer,        workers will realize that testing many different cell lines for        uptake in the case where cellular targeting is a subject of        scientific investigation, as intracellular uptake of many        bioactive molecules is novel and unanticipated from prior art in        the field of drug delivery;    -   7. Define, using microscopy, whether intracellular FITC-molecule        is contained in endosomes or it is free in cytoplasm; Typical        time points for imaging of endosomes is approximately 24 hr        after the initial exposure.    -   8. Define, using Western Blot expression of GLUT-transporters,        whether the intracellular action of molecule is expressed as        cell surface mediated uptake of additional substances or        molecules controlled by actions of intracellular molecule;    -   9. Prepare enteric coated pH 5.5 release capsule with        FITC-molecule-cholestosomes for administration to an animal or        human (the preferred oral administration form for acid labile        proteins, peptides, genes or live constructs such as vaccines or        viruses);    -   10. Administer oral dosage form of FITC-molecule-cholestosome to        mouse or human;    -   11. In the experiments of step 10, Administer same dose of        FITC-molecule-cholestosome orally as FITC-molecule-cholestosomes        in enteric coated capsule, IV; administer same dose of        FITC-molecule-cholestosome IV;    -   12. Compare effects on a biomarker of molecule effect after        administration of FITC-molecule-cholestosome between the three        modes:        -   a. oral as FITC molecule cholestosomes which result in            lymphatic chylomicrons loaded with FITC molecule            cholestosomes, vs.        -   b. Intravenously administered as FITC-molecule cholestosomes            which would not form chylomicrons and which may or may not            facilitate absorption of molecules into cells vs.        -   c. FITC-molecule intravenously and not in cholestosomes and            therefore not in chylomicrons) at the same dose of molecule            for each mode.    -   13. Using fluorescence microscopy, examine biodistribution of        FITC-molecule in tissues taken from mice given the 3 modes of        administration (a vs. b vs. c) in step 12 above. Tissues to be        examined post mortem include liver, kidney, brain, pancreas,        duodenum, ileum, colon, spleen, muscle, abdominal fat. It is        anticipated that high intracellular concentrations of molecules        can be achieved by this method, and that distribution in cells        would be uniform instead of confined to endosomes or digestive        vacuoles. Measurement of effect of molecule would be correlated        with intracellular distribution profile and a measure of overall        bioactivity vs. dose would be derived from the effect        measurements.

FIG. 12. Structure of a Cholestosome with Encapsulated Protein

Shown in FIG. 12 is a loaded cholestosome structural model withencapsulated insulin as an example. It is assumed that these ideal lipidparticles are aggregated into clumps of lipid, with raw production sizesof clumps of about 1000-5000 nm. Extrusion of these large particles downto uniformly sized 250 nm particles is a preferred embodiment. This canbe effected using a standard high pressure extrusion device, well knownin the art.

Example 2 Cholesteryl Ester Composition and Layering Preliminary Studiesof Cholesteryl Esters Considered for Use in Manufacture ofCholestosomes.

Define the melting point of each ester. By way of example, myristate hasa melt transition temperature of 65 degrees centigrade, above whichtemperature the solid component melts.

The formulation objective was to use cholesteryl esters at temperaturesbelow the melt temperature. (Consistent with liposome preparations), andconsidering that proteins begin to denature at temperatures about 40degrees centigrade.

Further temperature testing was carried out on the chosen estersmyristate and laurate. After the organic solvent was completely removedfrom the lipids in the rotovap, a DSC was conducted, which showed twomelting temperatures, one approximately 60 degrees centigrade and asecond melt at a higher Temperature.

On the basis of these findings and considering the stability of theproteins and peptides being formulated, the operating temperature ofencapsulation procedures was kept between 45 and 55 degrees centigrade.

Selection of Cholesteryl Esters and Compositions for Encapsulation ofMolecules in Cholestosomes

Selection of specific cholesteryl esters for the proper formation ofencapsulating vesicles involves a novel approach and a computerizedmolecular model. Properties of the cholesteryl esters and theinteraction between the target molecule for encapsulation and the innerhollow core of vesicle formed from the esters around the molecule can beused to define favorable cholestosome-molecule properties such asloading, either on a volume to volume basis or a weight to weight basis.

Cholestosome Vesicles prepared without molecules loaded inside, have anaverage diameter of 250 nm after extrusion. The size can be modified asa function of size of cholesteryl esters, mole ratios in mixtures ofdifferent cholesteryl esters, filtration techniques, sonication times,and temperature.

-   -   a. Cholesteryl esters claimed that form cholestosomes include:        Any cholesteryl ester produced from cholesterol and a fatty        acid, where a fatty acid includes both saturated and unsaturated        fatty acids including but not limited to the following compounds        in Table 2 below:

TABLE 2 Listing of fatty acids used to form cholesteryl esters,characterized by structure, ratio of Carbons to number of double bondsthe ratio C:D and position of the double bonds Position of Common doublename-Acid Chemical structure C:D bond MyristoleicCH₃(CH₂)₃CH═CH(CH₂)₇COOH 14:1 n-5 Palmitoleic CH₃(CH₂)₅CH═CH(CH₂)₇COOH16:1 n-7 Sapienic CH₃(CH₂)₈CH═CH(CH₂)₄COOH 16:1  n-10 OleicCH₃(CH₂)₇CH═CH(CH₂)₇COOH 18:1 n-9 Elaidic CH₃(CH₂)₇CH═CH(CH₂)₇COOH 18:1n-9 Vaccenic CH₃(CH₂)₅CH═CH(CH₂)₉COOH 18:1 n-7 LinoleicCH₃(CH₂)₄CH═CHCH₂CH═CH(CH₂)₇COOH 18:2 n-6 LinoelaidicCH₃(CH₂)₄CH═CHCH₂CH═CH(CH₂)₇COOH 18:2 n-6 α-LinolenicCH₃CH₂CH═CHCH₂CH═CHCH₂CH═CH(CH₂)₇ 18:3 n-3 COOH ArachidonicCH₃(CH₂)₄CH═CHCH₂CH═CHCH₂CH═CHCH₂ 20:4 n-6 CH═CH (CH₂)₃COOH^(NIST)Eicosapentaenoic CH₃CH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂ 20:5 n-3 CH═CHCH₂CH═CH(CH₂)₃COOH Erucic CH₃(CH₂)₇CH═CH(CH₂)₁₁COOH 22:1 n-9Docosahexaenoic CH₃CH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂ 22:6 n-3 CH═CHCH₂ CH═CHCH₂CH═CH(CH₂)₂COOH Caprylic CH₃(CH₂)₆COOH  8:0 Capric CH₃(CH₂)₈COOH 10:0Lauric CH₃(CH₂)₁₀COOH 12:0 Myristic CH₃(CH₂)₁₂COOH 14:0 PalmiticCH₃(CH₂)₁₄COOH 16:0 Stearic CH₃(CH₂)₁₆COOH 18:0 Arachidic CH₃(CH₂)₁₈COOH20:0 Behenic CH₃(CH₂)₂₀COOH 22:0 Lignoceric CH₃(CH₂)₂₂COOH 24:0 CeroticCH₃(CH₂)₂₄COOH 26:0

In the above table, C is the number of carbons and D is the number ofdouble bonds in the alkyl chain of the fatty acid molecule, C:D ratio ofthe molecule as displayed. The position of the double bond is expressedas the number of carbon after the carbonyl, which is position 1 in thechain. In this manner, n-5 for myristoleic acid means that the doublebond is found at position 14-5=position 9

The term “cholesterol” is used in the present invention to describe anycholesterol compound which may be used in the preparation of thecholesteryl esters which may be used to form cholestosomes pursuant tothe present invention. The term “cholesterol” and includes the moleculeidentified as cholesterol itself, and any related cholesterol moleculewith additional oxygenation sites (“an oxygenated analog ofcholesterol”) as in for example (but not limited to), 7-ketocholesterol,25-hydroxy cholesterol, 7-beta-hydroxycholesterol, cholesterol, 5-alpha,6-alpha epoxide, 4-beta hydroxycholesterol, 24-hydroxycholesterol,27-hydroxycholesterol, 24,25-epoxycholesterol. Oxysterols can vary inthe type (hydroperoxy, hydroxy, keto, epoxy), number and position of theoxygenated functions introduced and in the nature of theirstereochemistry. These various cholesterols may be used to providecholesterol esters which vary in solubility characteristics so as toprovide some flexibility in providing a cholestosome with a neutralsurface and groups which can instill hydrophilicity in the cholesterolester molecules. The cholesterol type molecule could also include anysterol structurally based compound containing the OH necessary for esterformation such as Vitamin D.

Molar ratios claimed in beneficial formation of cholestosomes range from0.05 to 0.95 of any pair of esters (when a pair of esters is used)listed in table 2 above. Product ratios of composition between pairs ofapproximately equal alkyl chain length cholesteryl esters and activemolecules range from about 2:2:96 to 48:48:4, often 45:45:10 to about2:2:96, about 40:40:20 to about 5:5:90, about 40:40:20 to about25:25:50. It is noted that in many cholestosome formulations when two(or more) cholesteryl esters are used, the ratio may vary above or belowa 1:1 ratio for the cholesteryl esters used.

Filtration techniques claimed include vacuum filtration for initial sizeselection and then extrusion of preparations for finer size selection.

Sonication times range from 30 min to 120 minutes. This time ispresented as a range, in that centrifuge time is a variable. Optimalsonication time depends on the ability to find the optimal sonicationspot in the sonicator, and at optimal timing, the solution forms acloudy appearance and the amount of solid material should be minimal asdetermined at this point by visual inspection.

Temperature range during production of cholestosome vesicles is 35° C.to 45° C. when working with most of the cholesteryl esters in Table 2.Temperature is held constant (+/−5 C) throughout the preparation of thevesicles. Temperature is kept below the melt temperature of any of theindividual esters. By way of example, for the preparation ofcholestosomes using myristate/laurate, temperature is held at 40° C.+/−5C. Addition of small amounts of between to the mixture prior tosonication increase overall yield of cholestosomes and facilitate theproduction of more uniform particles.

By means of example, the following principles define the basis forchoice of a component ester in a cholestosome, a means of choosing anester or ester pair for encapsulation purposes, and rely on thedisclosed physiochemical properties of the listed cholesteryl esters inTable 2:

-   -   1) The esters chosen for combination should be able to arrange        themselves to optimize the ester link interactions between ester        pairs. This electrostatic interaction is important for        orientation purposes, with the necessary hydrophobic exterior        and hydrophilic center of the vesicle.    -   2) The alkyl interactions should be able to optimize van der        Waals forces.    -   3) The sum of electrostatic interactions and the alkyl        interaction van der Waals forces are fundamental properties that        hold the vesicle shape and thereby retain the molecule inside. A        key additional factor for stability of cholestosome vesicles        includes the degree of repulsion between the dual hydrophobic        ends of the esters and the aqueous component containing the        molecule(s) to be encapsulated.    -   4) The overall size of the vesicle becomes a function of the        length of the alkyl chain. The increased length of the esters        chosen will increase the overall hydrophobic character of the        entire vesicle.    -   5) Using smaller chain length esters will actually increase the        overall hydrophilic character of the vesicle (in terms of the        overall structure of each ester).    -   6) Molecules that require more hydrophobic areas to assist in        encapsulation within the vesicle could benefit from esters        having longer alkyl chains.    -   7) Molecules that are smaller and require more hydrophilic        components to assist in encapsulation would benefit from ester        pairs that are shorter in length.    -   8) An additional choice is the use of unsaturated alkyl chains        such as those listed in Table 2, where these fatty acids are        used to prepare ester side chains for use in forming cholesteryl        esters.    -   9) The use of an unsaturated fatty acid offers an additional        structural modification in the vesicle structure which        incorporates additional electrostatic interactions between the        aqueous and the double bond character.    -   10) In the process of selection of esters for vesicle formation,        selection of CH₂ chain lengths ranging for example from 2 CH₂        units but less than 27 CH₂ in length result in a structure that        may not be as tight, as a result of the challenges in adapting        the alkyl chains to maximize their interactions in a vesicle.        The cholesterol component of the vesicle wall does not change.        The van der Waals interactions within CH₂ units governs the        flexibility of the alkyl interactions. However, for beneficial        hydrophilic vesicle center, the optimal configuration in this        vesicle is longer alkyl chains, meaning that larger ester        molecules have greater utility for stabilizing more hydrophilic        vesicle centers of the vesicle exposed to the aqueous        environment in formulation stability.

FIGS. 6-7 illustrate molecular modeling diagrams by means of an exampleof Cholestosome vesicle matrix formation from two different pairs ofcholesteryl esters selected from Table 2. In FIGS. 8-10 of themyristate-laurate matrix example chosen here, the inventors usedmolecular modeling to illustrate the impact on a small water solublemolecule Ceftaroline. The cholestosomes formed for Ceftaroline havecomposition ratios of 5:5:90 with the chosen pair of cholesteryl esters.In FIGS. 11-13, the representative peptide molecule was Insulin, apeptide of 6 kd size that is generally water soluble. In FIGS. 14-15,the cholestosome vesicle structure was applied to encapsulatebevacizumab, a representative monoclonal antibody of size approximately150 kd. In FIG. 16 all 3 representative molecules are shown in relationto the cholestosome vesicle formed from cholesteryl esters myristate andlaurate.

For ester pairs that are greater than 6 CH₂ units different in length(which is defined as intermediate) it is possible to maintain esterinteractions and turn the molecules in opposite directions to still havealkyl chains packed into a vesicle. This arrangement would be useful forpacking in molecules that have alternating structural regions ofhydrophobic/hydrophilic character, and which when incorporated into saidvesicle, could be relied upon to segregate different molecule types.

The choice of ester pairs is a function of the structure of the moleculeneeded to be encapsulated and its ability to interact with the vesicle.

In FIGS. 8-10, an outline structure of the entire cholestosome is shown,with inserted molecule ceftaroline. Insulin is shown in FIGS. 11-13, andin FIGS. 14-15 the molecule shown is bevacizumab. In FIG. 16, the matrixis shown around Ceftaroline, insulin and bevacizumab illustrated next toeach other, which nicely illustrates the relative sizes of thesemolecules in relationship to the size of the cholestosome matrix. Allthree of these molecules can be effectively encapsulated by thismethodology and used in humans after oral administration. In each casethere is no known effective means of oral absorption, other than asdisclosed in the present invention.

Example 3 Antibiotics Anti-Fungals, Anti-Virals and Other SmallMolecules in Cholestosomes for Oral Use

In the present invention, molecules used for the treatment of infectiousdiseases would be generally suitable for encapsulation intocholestosomes and used orally. Most antibiotics need to be injectedintravenously (IV), as the molecules are typically hydrophilic and nototherwise orally absorbed. Thus use in cholestosomes would make enabletheir oral absorption. Numerous antibiotics may be used in cholestosomesaccording to the present invention including Antibiotics for use in thepresent invention include Aminoglycosides, including Gentamicin,Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Spectinomycin;Ansamycins, including Geldanamycin, Herbimycin Rifaximin andStreptomycin; Carbapenems, including Ertapenem DoripenemImipenem/Cilastatin and Meropenem; Cephalosporins, including Cefadroxil,Cefazolin, Cephalothin, Cephalexin, Cefaclor, Cefamandole, Cefoxitin,Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, CefoperazoneCefotaxime Cefpodoxime, Ceftazadime, Ceftibuten, CeftizoximeCeftriaxone, Cefepime, Ceftaroline fosamil and Ceftobiprole;Glycopeptides, including Teicoplanin, Vancomycin and Telavancin;Lipopeptipdes, including Daptomycin, Oritavancin, WAP-8294A; Macrolides,including Azithromycin, Clarithromycin, Dirithromycin, Erythromycin,Roxithromycin, Telithromycin and Spiramycin; Lincosamides, includingClindamycin and Lincomycin; Monobactams, including Aztreonam;Nitrofurans, including Furazolidone and Nitrofurantoin; Oxazolidonones,including Linezolid, Posizolid, Radezolid and Torezolid; Penicillins,including Amoxicillin, Ampicillin, Azlocillin, Carbenicillin,Cloxacillin Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin,Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin,Temocillin and Ticarcillin; Penicillin combinations includingAmoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactamand Ticarcillin/clavulanate; Polypeptides, including Bacitracin,Colistin and Polymyxin B; Quinolones/fluoroquinolines, includingCiprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,Trovafloxacin, Grepafloxacin, and Sparfloxacin;

Sulfonamides, including Mafenide, Sulfacetamide, Sulfadiazine,Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfasalazine,Sulfisoxazole, Trimethoprim-Sulfamethoxazole and Sulfonamidochrysoidine;Tetracyclines, including Demeclocycline, Doxycycline, VibramycinMinocycline, Tigecycline, Oxytetracycline and Tetracycline;Anti-mycobacterial agents, including Clofazimine, Capreomycin,Cycloserine, Ethambutol, Rifampicin, Rifabutin, Rifapentine,Arsphenamine, Unclassified including Chloramphenicol, Fosfomycin,Fusidic acid, Metronidazole, Mupirocin, Platensimycin,Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline, Tinidazole andTrimethoprim.

None of these molecules are orally absorbed in the native state, and ineach case oral absorption would constitute a major advantage over thecurrent need to inject them parenterally in treatment of infectiousdiseases.

Examples of anti-fungal compounds for use in the practice of the art asapplied to cholestosome encapsulation include but are not limited to thefollowing miconazole, terconazole, econazole, isoconazole, tioconazole,bifonazole, clotrimazole, ketoconazole, butaconazole, itraconazole,oxiconazole, fenticonazole, nystain, naftifine, amphotericin B,zinoconazole and ciclopiroxolamine, micafungin, caspofungin,anidulafungin,

Examples of anti-viral compounds for use in the practice of the art asapplied to cholestosome encapsulation include but are not limited to thefollowing Ribavirin, telaprevir, daclatasvir, asunaprevir, boceprevir,sofosbuvir, BI201335, BI1335; ACH-2928, ACH1625; ALS-2158; ALS2200;BIT-225; BL-8020; Alisporivir; IDX19368; IDX184; IDX719; Simeprevir;BMS-790052; BMS-032; BMS-791325; ABT072; ABT333; TMC435; Danoprevir;VX222; mericitabine; MK-8742, GS-5885 or a mixture thereof, interferon,Pegylated Interferon, Pegylated interferon lambda or any other suitableformulation of said interferon.

Representative examples of anti-infective preparations in cholestosomesare disclosed herein, so as to illustrate the properties ofanti-infective substances in cholestosomes.

Tobramycin

A preferred embodiment illustrative of the molecules disclosed herein istobramycin, selected from this list for preparation and testing ofcholestosome encapsulated tobramycin according to the principlesenumerated in Example 1. The particular preparation was designed fororal use, and for increasing the overall action of the antibiotictobramycin against target gram negative bacteria such as Pseudomonasaeruginosa.

By way of specific example, tobramycin cholestosomes with mean diameterof 250-1,000 nm were prepared in the manner of the present invention, asdescribed in Example 1, with cholesteryl ester selection from the estersdisclosed as preferred in Example 2. Cholestosomes containing tobramycinwere prepared using a novel blend of two cholesteryl esters, cholesterylmyristate and cholesteryl laurate.

Tobramycin Formulation Properties Batch Properties

DLLS particle size: 2700 nm

Zeta Potential: −21.7

Concentration of Lipids: 1.9 mg/ml. Concentration of Tobramycin: 2.0mg/mlCell exposures: MCF-7 cells (See FIG. 23)Cholestosomes alone; No effect on growth or viability over 24 hrFITC alone: No effect on growth or viability over 24 hrTobramycin Alone: 10 mcg/ml to 0.01 mcg/ml No effect on growth orviability over 24 hrFITC Tobramycin alone; 10 mcg/ml No effect on growth or viability over24 hrFITC Tobramycin cholestosomes: 3.0 mcg/ml 24 hr killing, repeated, sameresult. Postulated 100× inside vs outside, with intracellular killingthreshold similar to renal tubular lining cells.Conclusions: Cholestosomes alone, FITC cholestosomes alone, Tobramycinalone do not kill MCF-7 cells. FITC-tobramycin on MCF-7 cells also doesnot harm them. However, FITC-tobramycin-cholestosomes kills at 24 hr.No chylomicron studies conducted with FITC tobramycin cholestosomes

FIG. 23 FITC Tobramycin Cholestosomes

Comparing MCF-7 cells by bright field vs FITC fluorescence imagingshows 1) an overall successful loading of MCF-7 cells after 24 hrexposure to FITC-cholestosomes, which has been shown repeatedly in ourwork with cholestosomes.

In 2), this response of approximately 100 fold greater concentration oftobramycin inside MCF-7 cells is unexpected, particularly when theloading of cells by cholestosomes is compared with the general lack ofintracellular loading of MCF-7 cells when exposed to FITC-tobramycinalone. Low loading is the expected result, as it is well known thattobramycin does not enter most body cells, and any cell that takes uptobramycin actively is subject to the intracellular killing fromtobramycin by virtue of its effect on mitochondria and cell energysupply via ATP production. This is the basis for tobramycin's well knownnephro and oto toxicity.

In 3) and of great interest, when MCF-7 cells were exposed toFITC-Tobramycin-cholestosomes for 24 hr, these MCF-7 cells all died, ascan be seen in the last frame at both top and bottom. The purpose hereis to show how tobramycin, when it enters cells, is a general toxin tothe mitochondria and when tobramycin enters even cells otherwiseresistant to its intracellular effects, there is potential forintracellular uptake and harm.

Ceftaroline

By way of a specific example concerning a cephalosporin antibiotic thatis not absorbed orally and is therefore currently given by IVadministration only, we chose the anti-MRSA cephalosporin antibioticCeftaroline fosamil.

Commercially available Ceftaroline was purchased from the hospitalpharmacy, and Ceftaroline cholestosomes were prepared in the manner ofthe present invention, as described in Example 1, with cholesteryl esterselection from the esters disclosed as preferred in Example 2. We wereunable to FITC label Ceftaroline, so the batches were tested for theirantimicrobial properties as the primary means of defining the efficacyof the formulation.

Test batches of cholestosomes containing Ceftaroline were prepared usinga novel blend of two cholesteryl esters, cholesteryl myristate andcholesteryl laurate. The choice of cholesteryl esters for composition ismade from the disclosed compounds of Example 2, although this is notmeant to be limiting and if there are other suitable cholesteryl estersfor formulation with ceftaroline or similar molecules, they may bepermitted in this formulation.

In the specific preparation of an optimal cholestosome formulationcontaining Ceftaroline, any cholesterol ester may be chosen as acomponent of the cholestosome and be within the spirit of the inventionso long as the final Zeta Potential of the cholestosome product remainsneutral charged.

Ceftaroline Formulation Properties Batch:

FITC label fraction: not doneDLLS particle size not done and not extrudedPreparation dialyzed to remove free Ceftaroline: yes, but freeCeftaroline remains in the preparationPercent yield 13% of starting amount of lipidZeta Potential: Not doneBacterial testing with the dialyzed Ceftaroline; Retains anti-MRSAaction, with MIC values at least 10× lower than parent Ceftaroline.Indicates active uptake by MRSA from cholestosome preparation.Cells: MCF-7; 400,000 cells at 24 hr in a confluent prep. MCF-7 cellSize is 2000 nmCholestosomes alone; No effect on MCF-7 cell growth or viability over 24hrFITC alone: No effect on MCF-7 cell growth or viability over 24 hrCeftaroline Alone: No effect on MCF-7 cell growth or viability over 24hrFITC ceftaroline alone; Not prepared so not doneFITC ceftaroline cholestosomes: No effect on MCF-7 cell growth orviability over 24 hrPostulate 100× inside vs outside.Chylomicron forming Cells: Ceftaroline was/was not tested in Caco-2cells

Vancomycin

By way of a specific example concerning a glycopeptide antibiotic thatis not absorbed orally and is therefore currently given by IVadministration only, we chose the anti-MRSA glycopeptides antibioticvancomycin.

Commercially available Vancomycin was purchased from Sigma chemical, andFITC vancomycin cholestosomes were prepared in the manner of the presentinvention, as described in Example 1, with cholesteryl ester selectionfrom the esters disclosed as preferred in Example 2. The batches werefully tested against MCF-7 cells, Caco-2 cells and also tested for theirantimicrobial properties against MRSA as the second primary means ofdefining the efficacy of the formulation.

Test batches of cholestosomes containing FITC-vancomycin were preparedusing a novel blend of two cholesteryl esters, cholesteryl myristate andcholesteryl laurate. The choice of cholesteryl esters for composition ismade from the disclosed compounds of Example 2, although this is notmeant to be limiting and if there are other suitable cholesteryl estersfor formulation with vancomycin or similar glycopeptides antibioticmolecules, they may be permitted in this formulation.

In the specific preparation of an optimal cholestosome formulationcontaining vancomycin, any cholesterol ester may be chosen as acomponent of the cholestosome and be within the spirit of the inventionso long as the final Zeta Potential of the cholestosome product remainsneutral charged.

Vancomycin Formulation Properties

Batch: 756, made 10-23-13DLLS particle size 1016 nm not extrudedDLLS particle size: 800 nm extrudedPreparation dialyzed to remove free vancomycinPercent yield <1.0% of starting amount of lipid

Zeta Potential: −13

Volume to Volume calculation:Concentration of Lipids: 1.0 mg in 10 ml. Concentration of Vancomycin:5000 mcg/mlWeight to Weight calculation:Concentration of Lipids: <1.0 mg/ml. There is free vanco in thispreparationBacterial testing with the dialyzed version of this, which killed MRSAvery well, vancomycin was approximately 10 times more active incholestosomes than used alone.Cells: MCF-7; 400,000 cells at 24 hr in a confluent preparation. MCF-7cell Size is 2000 nm.Cholestosomes alone; No effect for 24 hrFITC alone: No effect for 24 hrVancomycin Alone: no effect; up to 666 mcg/ml, highest testedFITC vanco alone; 666 mcg/ml to 41 mcg/ml: No effect for 24 hrFITC vanco cholestosomes: No effect at 24 hr. At a vancomycinconcentration of 0.83 mcg/ml from cholestosomes, FITC label study showsa very high internal vancomycin concentration in MCF-7 cells, equal tothe image labeling of 666 mcg/ml, see FIG. 24. From these data it ispossible to observe FITC-vancomycin concentrations 1000× inside vsoutside as the effect of cholestosome loading.Microbiological Activity against 4 different MRSA Strains: MIC values ofcholestosome vancomycin were equal to vancomycin or in some cases up to10× lower than vancomycin alone

FIG. 24. FITC Vancomycin Cholestosomes.

As shown in FIG. 24, vancomycin has some effective properties insidecells. This figure shows vancomycin entry into MCF-7 cells at 24 hr. Inthis series of experiments, the original starting concentrations ofvancomycin were between 41 and 666 mcg/ml. In each column, the top imageis the fluorescence, the bottom is the darkfield. Displayed out of thisFITC-vancomycin series in column B is FITC vancomycin at 83 mcg/ml. Incolumn A, FITC-vancomycin-cholestosomes at 0.83 mcg/ml produced greateruptake at a value 100 fold lower than the vancomycin concentration inFITC-vancomycin column B. The fluorescence image in column A shows moreloading than the image in column B, indicating that the MCF-7 cellularloading ratio is more than 100× greater withFITC-vancomycin-cholestosomes. When the extracellular concentration ofFITC-vancomycin was increased to 666 mcg/ml in column C, these cells arestill not loading as high as those in column A. The fluorescence data onloading of FITC vancomycin is therefore approaching 1000× greater whencholestosomes are used. It should be noted that there was no effect ofhigh amounts of FITC vancomycin cholestosomes on these MCF-7 cells. Theimages in the three panels confirm our observed penetration of FITCvancomycin cholestosomes inside cells. Not only are the cell membranesdramatically more concentrating FITC vancomycin in this image, but thecytoplasm of these cells is loaded with FITC vancomycin as well. This isafter only 24 hr exposure, confirming that cholestosomes load massivelymore vancomycin in the cells.

Chylomicron forming Cells: Vancomycin was not tested in Caco-2 cellsConclusion: Vancomycin alone, FITC vancomycin, FITC-vancomycincholestosomes, all at high concentrations, do not harm MCF-7 cells.Vancomycin retains its antimicrobial action on MRSA organisms whenencapsulated into cholestosomes.

Example 4 Insulin Cholestosomes Specific Steps in Preparation of Insulinin Cholestosomes.

By way of specific example, Regular Insulin (Humulin, Lilly)cholestosomes were prepared in the manner of the present invention, asdescribed in Example 1, with cholesteryl ester selection from the estersdisclosed as preferred in Example 2. Test batches of cholestosomescontaining insulin were prepared using a novel blend of two cholesterylesters, cholesteryl myristate and cholesteryl laurate. The choice ofcholesteryl esters for composition is made from the disclosed compoundsof Example 2, although this is not meant to be limiting and if there areother suitable cholesteryl esters for formulation with insulin orsimilar molecules, they may be permitted in this formulation.

In the specific preparation of an optimal cholestosome formulationcontaining insulin, any cholesterol ester may be chosen as a componentof the cholestosome and be within the spirit of the invention so long asthe final Zeta Potential of the cholestosome product remains neutralcharged. The two esters chosen for insulin using the principlesdisclosed in Example 2 were myristate and laurate, which differ in esterchain length by only two CH2 units, and when combined as disclosedprovide a large internal hydrophilic center to the cholestosome vesicleprepared in this manner.

Optimizing the amounts of specific cholesteryl esters is fully withinthe scope of the present invention for purposes of producing an optimalloading and release profile of the insulin containing cholestosome forin vivo use.

Initial starting conditions are based on a 1:1 molar ratio oflaurate/myristate, while the final ratio in the formulation of thevarious insulin molecules is not limited to that. Each insulin moleculewill need to be examined in terms of its own structure and the molecularinteractions with the putative cholesteryl esters as a means of finalselection of cholesteryl esters for optimal loading. In the event theoptimal final formulation requires a more hydrophobic area, then alonger chain fatty acid ester is used, as the entire proportion ofhydrophobic space will change based on the length of the alkyl chain. Ifwe need more centralized hydrophilic structures for certain insulinmolecules, the intention is to use one of the oxysterols such as 7-ketocholesterol made into an ester with fatty acids.

The encapsulation molecule is insulin, to include but not limited toregular insulin, NPN insulin, insulin glargine, insulin degludec or anyformulation of insulin prepared and shown to be bioactive in testing forinsulin effects. Steps in preparation of the cholestosome formulationincluded the following:

Prepare a water bath to appropriate temperature (35-45) C; Place aqueousinsulin prep (1 mg/ml) in PBS into water bath to equilibratetemperature; Weigh out equimolar amounts of cholesteryl laurate andcholesteryl myristate (75 mg each) and place in round bottom flask; Addorganic solvent (diethyl-ether) to dissolve esters; swirl by hand todissolve; Place round bottom flask on rotovap and spin for five minutes;Place flask attached to rotovap in water bath; turn on vacuum and spinfor 10 minutes; Turn off rotovap and vacuum and add aqueous to roundbottom flask; Add Tween; Spin on rotovap (no vacuum) for twenty minutesin water bath; Sonicate for 10 to 30 minutes until cloudy prep is formedand minimum solid is found in flask; Remove from sonication and filterusing vacuum filtration; Save the cloudy filtrate; Extrude filtrate;Store preparation in refrigerator until use.

FIG. 17 Caco-2 Studies in Transwell; Formation of Chylomicrons

Employ Corning Transwell Permeable Supports in a 12 well format with apore size of 0.4 um. Begin each Transwell experiment after Caco2 cellsare 80-90% confluent in a 75 cm² flask. The cells are trypsinized asusual and counted using a hemocytometer. The cell concentration isadjusted to 2×10⁵ cells/mL with culture media. The wells of theTranswell plate are seeded with 0.5 mL of the cell dilution. Media in avolume of 1.5 ml is added to the basolateral side. The cells areincubated as above and the media is changed every other day for 19-20days. At this time the caco2 cells are differentiated and ready fortreatment. All media from the upper and lower chambers of the Transwellplate is removed and both chambers are washed 3 times with PBScontaining 1 mg/mL glucose (PBSG). PBSG is added to the upper and lowerchamber of the plate and incubated for 1 hr. All PBSG is removed fromboth chambers and 1.5 mL of phosphate buffered saline with added glucose(PBSG) is added to the lower chamber.

The upper chamber receives 0.5 mL of the appropriate treatment (PBSGalone, FITC cholestosomes in PBSG or FITC-insulin cholestosomes inPBSG). All wells have a final concentration of 1.0 mg/mL glucose. Theplate is then incubated for 2 hours. All solution is removed and viewedon the Zeiss confocal LSM 510 microscope.

FIG. 18 is an image of the apical side of the Transwell plate at 1 hrafter exposure to FITC labeled insulin (i.e. not in a cholestosome)

In FIG. 19, the basolateral fluid is imaged on the confocal microscope.In this case the apical side had only PBS buffer and media (no FITC, noinsulin, no cholestosomes) There is no visible fluorescence and thisimage represents image background for the basolateral images to follow.

In FIG. 20, the image is following FITC cholestosomes applied to theapical side for 2 hr, which shows small chylomicrons containing FITC inthe basolateral fluid. It is important to note that this fluid wasimaged after collection of the basolateral fluid and does not reflectmicroscopy across the entire preparation. Hence, these chylomicrons wereclearly formed by the Caco2 cells.

In FIG. 21, the image is following FITC insulin cholestosomes applied tothe apical side for 2 hr, which shows overall larger chylomicronscontaining FITC-insulin in the basolateral fluid. It is important tonote that this fluid was imaged after collection of the basolateralfluid and does not reflect microscopy across the entire preparation.Hence, these FITC insulin containing chylomicrons were clearly formed bythe Caco2 cells.

Summary of Cholestosome Insulin Formulation Properties

Batch: 733 and pooled batchDLLS particle size 1700 nm not extrudedDLLS particle size: 149-274 nm after extrusionPercent yield 13.0% of starting amount of lipid

Zeta Potential: −24.7

Loading ratio: Loading weight to weight for regular insulin was 13%insulin to 87% sum of cholesteryl myristate plus cholesteryl laurate.Cells: MCF-7; n=400,000 cells at 24 hr in a confluent preparation; Sizeis 2000 nmCholestosomes alone; No effect on growth or viability over 24 hrFITC alone: No effect on growth or viability over 24 hrInsulin Alone: 3 mcg/ml of 1.5 ml volume; (4.5 mcg) No effect for 24 hrFITC Insulin alone; 466 mcg/ml No effect on growth or viability over 24hr (see FIG. 22)FITC Insulin cholestosomes: 0.46 mcg/ml; No effect on growth orviability over 24 hrInsulin uptake starting by 2 hrs. (see FIG. 22)FITC Insulin cholestosome chylomicrons: Massive uptake with all cellmembranes engaged at 2 hr, free insulin in cytoplasm. Concentrationinside MCF-7 cells at least 1000× over concentration outside cells.

Cells: Caco-2

Concentration apical side: Pre: 350 ul of 0.46 mcg/ml cholestosomesolution on apicalFITC Cholestosomes alone; No effect on Caco-2 cells over 24 hr;chylomicrons formed as in FIG. 20FITC insulin alone; No effect on Caco-2 cells over 24 hr; nochylomicrons formed on basolateral side as in FIG. 18FITC alone: No effect on Caco-2 cells for 24 hr; no Chylomicrons onBasolateral side (FIG. 19)Insulin Cholestosomes: 0.46 mcg/ml with free insulin-transferred allcholestosomes to basal side as chylomicrons (FIG. 21)

Chylomicrons formed with FITC Insulin cholestosomes: Insulinconcentration 0.46 mcg/ml or lower. (FIG. 22)

FIG. 22: FITC Insulin Cholestosome Chylomicrons

FIG. 22. The original starting concentration for FITC-insulin exposurein MCF-7 cells was 466 mcg/ml, which did not result in measurableamounts of FITC insulin inside the MCF-7 cells in row A. For the twolower figures (rows B and C), the concentration of FITC insulincholestosome was 0.46 mcg/ml, which is the same for the experimentssummarized in the last 2 figures. The 0.46 mcg/ml from FITC insulincholestosomes (row B) produced about the same intracellular fluorescenceas 466 mcg/ml of FITC insulin without cholestosomes (row A). Compared to466 mcg/ml of FITC insulin without cholestosomes (row A), the furtherprocessing of FITC insulin cholestosomes by Caco-2 cells intochylomicrons, produced a robust improvement in the amount of insulininside cells from FITC insulin cholestosome-chylomicrons (row C), muchgreater than 1000 fold over the amount of FITC-insulin alone, and muchgreater than the effect of the 0.46 mcg/ml of insulin when not processedby Caco-2 cells. Assuming the amount passing Caco2 cells was all of theinsulin administered to the apical side, the concentration of insulin inthe FITC insulin cholestosome chylomicron row C was the same as theinsulin concentration in the middle row Row B. This particularpreparation had free insulin remaining, and if transfer across Caco-2cells was less than 100%, then these intracellular loading ratios areeven greater. Clearly, FITC insulin cholestosome-chylomicrons achievesgreater loading inside the cells, once again demonstrating thatcholestosomes alone do allow peptides to enter cells across the cellmembrane, as was earlier shown with FITC cholestosomes alone. The imagein the bottom row C reflects the observed penetration of FITC insulincholestosome chylomicrons inside cells. Not only are the cell membranesdramatically more concentrating FITC insulin in this image, but thecytoplasm of these cells is loaded with FITC insulin as well. This isafter only 2 hr exposure, confirming that chylomicrons not only loadmassively more, they load more quickly than cholestosomes on their own.

FIG. 25 FITC Insulin Cholestosome Chylomicrons Loading MCF-7 Cells

Cholestosomes containing encapsulated FITC-insulin were prepared asdisclosed herein, using FITC labeled regular insulin purchasedcommercially. Caco-2 cells were used to ensure that Cholestosomestransfer intact insulin (i.e. insulin remains within the Cholestosome)across the enterocytes and enters chylomicrons, following whichchylomicrons were detected on the basolateral side of the Caco-2membrane. ELISA was used to demonstrate that acid protected insulin doesnot pass the apical Caco-2 barrier (<5%), and that all of the insulin onthe basolateral side is within chylomicrons. FITC-insulin was used onthe apical side to verify that insulin alone does not pass theenterocyte barrier but that FITC insulin in cholestosomes passes theCaco2 enterocyte barrier. From these experiments, absorption efficiencywas determined as the difference between basolateral side and apicalside content of insulin. Further experiments compared the effect ofaltered pH and bile salts on the cholestosome encapsulated insulin. Inaddition, chylomicron stability when containing insulin loaded intocholestosomes was quantified and the conditions necessary for release ofinsulin from the loaded cholestosomes in vivo were studied.

In FIG. 25, the chylomicrons loaded with FITC insulin cholestosomes wereplaced adjacent to MCF-7 cells in order to demonstrate uptake intocells. These cells readily incorporate cholestosomes, and it appearedthat distribution was uniform within the cell.

In this experiment FITC insulin cholestosome chylomicron loading ofMCF-7 cells was improved over some of our previous experiments with FITCinsulin cholestosomes, and here the loading was 1000× greater from FITCinsulin cholestosome chylomicrons. In all cases, processing of FITCinsulin cholestosomes by Caco-2 cells into chylomicrons, produces arobust improvement in the amount of insulin inside cells from FITCinsulin cholestosome-chylomicrons (row B), Not only are the cellmembranes dramatically more concentrating FITC insulin in this image,but the cytoplasm of these cells is loaded with FITC insulin as well.This is after only 2 hr exposure, confirming that chylomicrons not onlyload massively more, they load more quickly than cholestosomes on theirown.

This particular formulation was administered to 4 mice.

FIG. 26. FITC Insulin Cholestosomes Given to 4 Mice.

Following completion of the in vitro studies in Caco-2 cells and MCF-7cells, the cholestosome insulin formulations could be administered tomice; ELISA is used to define insulin absorption and release fromchylomicrons and as a means of defining the biological residence ofinsulin circulating in cholestosomes in vivo.

Blood glucose is measured in the mice to define the effect of insulin inthe mouse model after administration of the formulations.

FIG. 26 shows the blood glucose values of four mice, each givenFITC-insulin-cholestosomes orally, with subsequent 30 minute glucosemeasurements using a glucometer. All mice tolerated the procedures well.In 3 of the 4 animals, the drop in blood glucose followed oraladministration by 30-45 minutes. In the fourth animal, the drop in bloodglucose began after 2 hrs, but had a similar decline and recovery time.In all cases, the glucose returned to baseline rapidly.

Overall, these data show oral insulin absorption and systemic effects onblood glucose, a demonstration of proof of concept for the cholestosomeformulations in a murine model.

Example 5 Cholestosomes Encapsulating Anti-Cancer Agents

The use of small and large molecules in the treatment of cancer is oftenlimited by barriers that need to be crossed in order to reach targetsites inside the cell. Inventors and specialists have long sought ameans of delivering small and large molecules across the cell membranebarrier, as a means of treating cancers of all types.

Thus the use of cholestosomes to promote oral absorption of anti-canceragents and enable distribution to intracellular pathways of molecularinteraction with cellular processes is of great interest, as most of themolecules to be listed below have intracellular delivery problems, oralabsorption problems, or both.

Described herein is a preferred embodiment of oral delivery andintracellular loading of anti-cancer molecules using endogenously formedchylomicrons. For the most part, the listed anti-cancer agents disclosedin this example are not proteins, genetic material or the like. Theseare considered small molecules, and the choice of a group of smallmolecules active against cancer should not be considered limiting, assmall molecules in general will follow the principles of encapsulationand oral absorption and intracellular uptake described herein. In allcases, one skilled in the art that pertains to the present inventionwill understand that there are equivalent alternative embodiments, theimportant feature of the present invention being reliable oralabsorption and intracellular delivery of the molecule in an intact form.In each of these representative cases, the molecule will be encapsulatedusing the methods disclosed in example 1 and example 2, developed andtested using similar models and processes defined for antibiotics inExample 3. These methods are not limiting and physical properties ofsome of the representative molecules given in this example may define apathway outside the boundaries of the Examples heretofore. As such,these will remain in the spirit of the invention.

Preferred Anti-Cancer Agents for Cholestosome Encapsulation

Representative anti-cancer molecules might include 5-Azacytidine;Alitretinoin; Altretamine; Azathioprine; Amifostine; Amsacrine;Anagrelide; Asparaginase; N-(phosphonyl)L-aspartic acid; Bexarotene(Targretin); Bleomycin; Bryostatin; Busulfan; Capecitabine;Camptothecin; Carboplatin; Carmustine; Carboprost (CarboprostTromethamine); Carglumic Acid; Carmofur; Chlorambucil; Cladribine;Clofarabine; Clofazimine; Colchicine; Curcumin; Difluorinated Curcumin(CDF); Cyclophosphamide; Cytarabine; Cytosine arabinoside;D-Aminolevulinic Acid; Dacarbazine; Daunorubicin/Daunomycin;Deferasirox; Denileukin diftitox (Ontak); Docetaxel/Taxotere;Doxifluridine; Doxorubicin/Adriamycin; Eflornithine; Epirubicin;Elephantopin; Estramustine; Etoposide Phosphate; Fludarabine;Fluorouracil; fluoroorotic acid; Fotemustine; Gemcitabine; Gusperimus;Hydroxycarbamide; Hydroxyurea; Idarubicin/4-Demethoxy Daunorubicin;Ifosfamide; Incadronate; Irinotecan; Peg-Irinotecan; Lapatinib/LapatinibDitosylate; Lomustine; Masoprocol; Melphalan Hcl; Mercaptopurine;Methotrexate (Amethopterin); Methyl Aminolevulinate; Mitomycin;Mitotane; Mitoxantrone; Nimustine Hydrochloride;Octadecylphosphocholine; Ormaplatin; Oxaliplatin; Paclitaxel;Peg-asparaginase; Pemetrexed; Pentostatin/Deoxycoformycin; PorfimerSodium; Procarbazine; Protein Kinase C inhibitors; Raltitrexed;Phenylbutyrate Sodium; Staurosporine; Streptozocin; Tafluposide;Temozolomide; Teniposide; Thioguanine; Thiotepa; Thymopoietin;Tioguanine; Tomudex; Topotecan; Tretinoin; Tropisetron hydrochloride;Uramustine (Uracil Mustard); Valrubicin; Verteporfin; Vinblastine;Vincristine; Vindesine; Vinorelbine; Vorinostat;

Curcumin Di-Fluoride (CDF) Example

The preferred embodiment illustrative of the molecules disclosed hereinis a derivative of curcumin, curcumin di-fluoride which is also calledCDF. Beneficial anti-cancer properties of CDF are well described in theart (16, 85-92). One of the actions of CDF is upon the histonemethyltransferase EZH2, which is a central epigenetic regulator of cellsurvival, proliferation, and cancer stem cell (CSC) function. EZH2expression is increased in various human cancers, including highlyaggressive pancreatic cancers, but the mechanisms underlying for itsbiologic effects are not yet well understood. The authors probed EZH2function in pancreatic cancer using diflourinated-curcumin (CDF), anovel analogue of the turmeric spice component curcumin that hasantioxidant properties. CDF decreased pancreatic cancer cell survival,clonogenicity, formation of pancreatospheres, invasive cell migration,and CSC function in human pancreatic cancer cells. These effects wereassociated with decreased expression of EZH2 and increased expression ofa panel of tumor-suppressive microRNAs (miRNA), including let-7a, b, c,d, miR-26a, miR-101, miR-146a, and miR-200b, c that are typically lostin pancreatic cancer. Mechanistic investigations revealed thatre-expression of miR-101 was sufficient to limit the expression of EZH2and the proinvasive cell surface adhesion molecule EpCAM. In anorthotopic xenograft model of human pancreatic cancer, administration ofCDF inhibited tumor growth in a manner associated with reducedexpression of EZH2, Notch-1, CD44, EpCAM, and Nanog and increasedexpression of let-7, miR-26a, and miR-101. Taken together, the resultsindicated that CDF inhibited pancreatic cancer tumor growth andaggressiveness by targeting an EZH2-miRNA regulatory circuit forepigenetically controlled gene expression.(89)

Cholestosome encapsulated CDF can be prepared for testing according toexample 1 procedures. FITC labeled CDF was used to assessbiodistribution, and the aforementioned epigenetic pathways were studiedwhen exposed to CDF cholestosomes before passage thru caco2 cells andthen after, when collected chylomicrons were used in the cellularsignaling pathway experiments.

Following the conclusion of the in vitro and cellular distributionexperiments, the CDF cholestosomes can be applied to a mouse model forassessment of intracellular exposure and action, in order to defineconcentrations and dosage vs bioactivity, with un-encapsulated CDF usedas a control. Both oral and IV administration were performed to definebioavailability as well as cellular uptake and localization. A secondpreferred embodiment is doxorubicin, itself a molecule oftenincorporated into liposomal drug delivery systems and widely used in thetreatment of cancer

Example 6 Bevacizumab as Representative of Monoclonal Antibodies

Described herein is a preferred embodiment of oral delivery ofmacromolecules to include peptides, proteins including monoclonalantibodies, genetic material or the like. These are considered largebiological molecules with molecular weight in excess of 6 kd and mostfrequently in excess of 100 kd, and the choice of a group of largebiomolecules active against diseases should not be considered limitinguse of the invention to a particular disease or treatment, asbiomolecules in general will follow the principles of encapsulation andoral absorption and intracellular uptake described herein. In all cases,one skilled in the art that pertains to the present invention willunderstand that there are equivalent alternative embodiments, theimportant feature of the present invention being reliable oralabsorption and intracellular delivery of the biomolecule in an intactform for the treatment of disease in human patients in the field ofprotein therapeutics. The monoclonal antibodies bevacizumab andtrastuzumab have been the principle subjects of encapsulation, but theseshould not be considered limiting and in fact most monoclonalantibodies, being of similar length, charge and molecular weight, willbehave similarly with respect to cholestosome encapsulation as describedherein.

Bevacizumab in Cholestosomes

A preferred embodiment illustrative of the molecules disclosed herein isbevacizumab, selected from this list for preparation and testing ofcholestosome encapsulated bevacizumab according to the principlesenumerated in Example 1. The particular preparation was designed fororal use and intracellular delivery, and corresponding IV use fortargeting of cell surface receptor target sites.

By way of specific example, bevacizumab cholestosomes with mean diameterof 250-10,000 nm can be prepared in the manner of the present invention,as described in Example 1, with cholesteryl ester selection from theesters disclosed as preferred in Example 2. Cholestosomes containingbevacizumab were prepared using a novel blend of two cholesteryl esters.

Alternative formulations of bevacizumab, as nanoparticles can beprepared as disclosed by Woitiski(6). These nanoparticles will bealbumin coated for Caco-2 experiments, to enable what is anticipated tobe maximal absorption capability, since coating improved the absorptionof insulin in this particular nanoparticle formulation.

Loading and Cellular Uptake with Bevacizumab Cholestosomes.

The formulation protein bevacizumab was labeled with FITC prior toincorporation into cholestosomes in a manner described in example 1.

Cholestosome loading with Bevacizumab on a weight to weight basis wasapproximately 20% in particles ranging in size from 250-10,000 nm.

All formulations will be examined using confocal microscopy, scanningelectron microscopy (SEM) and transwell experiments as disclosed by theinventors for insulin.

Caco-2 Cells for Testing Bevacizumab Cholestosomes

The Caco-2 cells used for the transwell experiments are cultured at 37°C. in an atmosphere of 5% CO²/95% O² and 90% relative humidity inDulbecco's Modified Eagle Medium (DMEM) supplemented with 100 IU/mLpenicillin and 100 mcg/mL streptomycin, 2 mM 1-glutamine, 1%non-essential amino acids, and 10% heat inactivated fetal bovine serum.Caco-2 cells form an absorptive polarized monolayer, and develop anapical brush border and secrete enzymes after culture for 21 days.

In addition to inspection by microscopy, trans-epithelial electricalresistance is measured across cells growing on 1 cm² polycarbonatefilters of trans-well diffusion cells using an epithelial volt ohmmeterto evaluate tight junctions.

Cholestosomes containing encapsulated FITC-bevacizumab were prepared asdisclosed herein, using FITC labeled bevacizumab purchased commercially.Caco-2 cells were used to ensure that Cholestosomes transfer intactbevacizumab (i.e. bevacizumab remains within the Cholestosome) acrossthe enterocytes and enters chylomicrons, following which chylomicronswere detected on the basolateral side of the Caco2 membrane. Fluorescentreadings of the FITC-bevacizumab preparation were used to demonstratethat free bevacizumab does not pass the apical Caco-2 barrier (<5%), andthat much of the FITC-bevacizumab placed on the apical side encapsulatedin cholestosomes was actually transferred to the basal side aschylomicrons containing the FITC-bevacizumab-cholestosomes.

Based on fluorescent readings, 75% of the FITC-bevacizumab-cholestosomesadded to the apical side of the Caco-2 enterocyte barrier passes theCaco2 enterocyte barrier. From these experiments, absorption efficiencywas determined as the difference between basolateral side and apicalside content of FITC-bevacizumab-cholestosomes. At the end of theexperiment at 24 hrs, all of the fluorescence readings added up to thestarting amount of fluorescence of the FITC-bevacizumab-cholestosomes,thereby achieving mass balance in the experiment itself.

MCF-7 Cell Experiments for Bevacizumab Cholestosomes and Bevacizumabcholestosome Chylomicrons

MCF-7 cells readily take up cholestosomes as shown in FIGS. 5, 22 and 23all control experiments for the respective constructs. Others are notshown herein. Because this is a fluorescent image, the only content ofthe cholestosomes in this image was from FITC encapsulated in thecholestosomes. Note the outline of the cell membrane which is loaded bycholestosomes, and in later images the uniform distribution of FITClabel throughout the cell and even to include its nucleus.

MCF-7 cells are relatively resistant to bevacizumab when subjected toin-vitro testing, having an IC50 value approximately 1.0 mcg/ml. Indeedthe drug functions indirectly as a cytostatic agent, which is the neteffect of blocking VEGF and decreasing the supply of blood vessels togrowing tumors.

Entirely expected based on the aforementioned in-vitro resistance, MCF-7cells show no uptake of FITC-bevacizumab at external concentrations of173 mcg/ml, a concentration approximately 10 fold higher than thetypical peak when a dose of 100 mg is given to a human under treatmentfor carcinoma. These data are part of FIG. 27.

These same MCF-7 cells were then exposed to FITC-bevacizumabcholestosomes, prepared according to the methods in Example 1, usingmyristate and laurate cholesteryl esters. These cholestosomes wereapproximately 5000-10,000 nm in size, while an MCF-7 cell isapproximately 15,000 nm in size. Both darkfield and fluorescent imagesof these MCF-7 cells were taken for 24 hr, and displayed in FIG. 28.These cells do not measurably take up bevacizumab cholestosomes, and itappeared that FITC-bevacizumab distribution was uniform within the cell.The MCF-7 cells remained viable for 24 hrs in this experiment,indicating that FITC bevacizumab-cholestosomes did not increase theaction of bevacizumab on these cells.

The same preparation of bevacizumab-FITC-cholestosomes was then exposedto Caco-2 cells, and the resulting chylomicrons containingFITC-bevacizumab-cholestosomes were collected from the transwellbasolateral side after 24 hr exposure. In this experiment, 75% of theBevacizumab-FITC-cholestosomes passed the Caco-2 barrier and wereincorporated into the resulting chylomicrons.

Because 75% of the cholestosomes were inside the chylomicrons, the MCF-7cells were exposed to a bevacizumab concentration similar to theconcentration of bevacizumab in the cholestosome preparation shownearlier. Of interest, the uptake into the MCF-7 cells was dramaticallygreater when chylomicrons were used for intracellular delivery ofFITC-bevacizumab-cholestosomes than when delivery was from cholestosomesalone or indeed from just exposing MCF-7 cells to free bevacizumab.

Furthermore, the MCF-7 cells exposed to chylomicron delivered FITCbevacizumab were non-viable in as little time as 4 hr after exposure.This is very remarkable because there is no known cytotoxic component tothe mechanism of action of bevacizumab. Heretofore, this Monoclonalantibody has a cytostatic mechanism the functions indirectly of VEGF andblood vessel growth. Furthermore, as bevacizumab is unable to entercells, the unexpected discovery of a rapid cytotoxic pathway fromintracellular delivery creates a new product and a new pathway for thisold protein.

Bevacizumab Formulation Properties

-   -   Date of manufacture: Aug. 3, 2013    -   DLLS particle size 10,510 nm; not extruded    -   Percent yield 20% of starting amount of lipid    -   Zeta Potential: Not done for bevacizumab. Trastuzumab: 6.4    -   Cells: MCF-7; 400,000 cells at 24 hr in a confluent prep. MCF-7        cell. Size is 2000 nm    -   MCF-7 cells with Cholestosomes alone; No effect on growth over        24 hr    -   FITC alone: No effect on growth 24 hr    -   Bevacizumab Alone: not tested    -   FITC bevacizumab alone; up to 173 mcg/ml: no effect on growth        over 24 hr (FIG. 27)    -   FITC bevacizumab cholestosomes: ˜20 mcg/ml; Well tolerated by        cells; visible intracellular uptake starting by 2 hrs.    -   FITC bevacizumab cholestosome chylomicrons from Caco-2 cells: 15        mcg/ml on MCF-7 cells for 4 hr with complete killing of all        cells in field. (FIG. 28)

FIG. 27. FITC Bevacizumab on MCF-7 Cells

FIG. 27 shows the dark field (top row) and fluorescent images at 2 hr, 4hr and 24 hr from the application of a target concentration of 173mcg/ml of FITC Bevacizumab to MCF-7 cells. These concentrations are 5-10fold greater than typically observed in Bevacizumab treated patients.There was no evidence that FITC bevacizumab integrated with the cellmembranes of these MCF-7 cells. There was no evidence of anyfluorescence uptake of FITC bevacizumab at any time point by MCF-7cells, and there was no evidence of effect of FITC-bevacizumab on theseMCF-7 cells.

FIG. 28 FITC Bevacizumab Cholestosome Chylomicrons Kills MCF-7 Cells

FIG. 28. In this experiment, FITC bevacizumab cholestosomes wereprepared and tested against MCF-7 cells. There was no effect at 2 hr,and the cells showed no uptake of FITC bevacizumab cholestosomes. Thenthese same FITC bevacizumab cholestosomes were placed on the apical sideof the Caco-2 cells and the resulting FITC bevacizumab cholestosomechylomicrons were tested on MCF-7 cells. The first frame of the bottomrow shows massive uptake of FITC bevacizumab cholestosome chylomicrons,and the other intensely observed effect was rapid cellular killing ofthe MCF-7 cells, as they were all killed by 4 hrs into the experiment.

Representative Monoclonal Antibodies and Large Proteins

Representative macromolecules for conversion to oral use or for improvedaction inside cells by use of the present invention might include anyone or combinations of those listed here, and include similar sized andcharged molecules that are discovered after disclosure of the compoundslisted herein: Adalimumab (Humira); Abciximab; Alemtuzumab; Bevacizumab,(Avastin); Bapineuzumab; Cetuximab; Etanercept, (Enbrel); Elotuzumab;Gemtuzumab; Inotuzumab; Kynamro™ mipomersen by Isis-Genzyme;MabThera/Rituxan; Natalizumab, Tysabri by Elan/Biogen; Necitumumab byEli Lilly; Palivizumab (Synagis); Panitumumab; RN316 (anti-PCSK9 byPfizer) REGN727 (anti-PCSK9 by regeneron) for lowering LDL cholesterol;Solanezumab; Trastuzumab (Herceptin); Tositumomab; T-DM1, an antibodydrug conjugate by Roche/Genentech, which consists of trastuzumab(Herceptin), DM1 (emtansine) and a linker that joins DM1 to trastuzumab;T-DM1 is designed to target and inhibit HER2 signaling and deliver DM1directly inside HER2-positive cancer cells; Zelboraf® for BRAF V600mutation-positive metastatic melanoma; Atorolimumab; Belimumab;Brodalumab; Carlumab; Dupilumab; Fresolimumab; Golimumab; Lerdelimumab;Lirilumab; vrilimumab; Metelimumab; Morolimumab; Namilumab; Oxelumab;Placulumab; Sarilumab; Sifalimumab; Tabalumab; Ipilimumab; Tremelimumab;Nivolumab; Urelumab; Bertilimumab; Zanolimumab; Afelimomab; Elsilimomab;Faralimomab; Gavilimomab; Inolimomab; Maslimomab; Nerelimomab;Odulimomab; Telimomab; Vepalimomab; Zolimomab aritox; Basiliximab;Clenoliximab; Galiximab; Gomiliximab; Infliximab (Remicade by Janssen);Keliximab; Lumiliximab; Priliximab; Teneliximab; Vapaliximab;Aselizumab; Apolizumab; Benralizumab; Cedelizumab; Certolizumab pegol;Daclizumab; Eculizumab; Efalizumab; Epratuzumab; Erlizumab; Etrolizumab;Fontolizumab; Itolizurnab; Lampalizumab; Ligelizumab; Mepolizumab;Mogamulizumab; Natalizumab; Ocrelizumab; Ofatumumab; Omalizumab;Ozoralizumab; Pascolizumab; Pateclizumab; Pexelizumab; Pidilizumab;Reslizumab; Rontalizumab; Rovelizumab; Ruplizumab; Quilizumab;Samalizumab; Siplizumab; Talizumab; Teplizumab; Tocilizumab;Toralizumab; Tregalizumab; Vatelizumab; Vedolizumab; Visilizumab;Ibalizumab; Otelixizumab; Briakinumab; Canakinumab; Fezakinumab;Secukinumab; Sirukumab; Tralokinumab; Ustekinumab; Anrukinzumab;Clazakizumab; Enokizumab; Gevokizumab; Ixekizumab; Lebrikizumab;Olokizumab; Perakizumab; Tildrakizumab; Besilesomab; Fanolesomab;Lemalesomab; Sulesomab;

Example 7 Oral Universal Lipid Control Combination Antibody to PCSK-9with Brake and Statin

A preferred embodiment illustrative of the molecules disclosed herein isAlirocumab, also known as REGN727, a monoclonal antibody against PCSK-9.Alternative monoclonal antibodies against PCSK-9 include or Evolocumabor Bococizumab by way of non-limiting example.

Alirocumab, selected from this list for preparation and testing ofcholestosome encapsulated antibodies to PCSK-9 according to theprinciples enumerated in Example 1. The particular preparation wasdesigned for oral use and intracellular delivery, upon knowledge andbelief that PCSK-9 is an intracellular target for an antibody againstthis compound.

By way of specific example, Alirocumab cholestosomes with mean diameterof 250-10,000 nm can be prepared in the manner of the present invention,as described in Example 1, with cholesteryl ester selection from theesters disclosed as preferred in Example 2. Cholestosomes containingAlirocumab are prepared using a novel blend of two cholesteryl esters.

In the treatment of hyperlipidemia, it is necessary to controlcholesterol, which is defined in clinical guidelines as raising HDL andlowering LDL, and in addition it is necessary to lower plasmatriglycerides. The oral combination product disclosed in this examplewill be the only available means of complete control of all aspects ofhyperlipidemia, and in addition it will remove the major disadvantage ofall members of the PCSK-9 monoclonal antibody treatments, the need forbi-weekly subcutaneous injection. Oral administration of PCSK-9monoclonal antibodies will significantly improve patient acceptance ofthese new therapeutic modalities.

PCSK-9 Monoclonal Antibody Component of Combination Product

Specific to the proteins of therapeutic benefit disclosed in exampleOral formulation of a monoclonal antibody to PCSK9 will control elevatedLDL in a potent manner, and the selected protein for oral cholestosomeencapsulation.

A preferred embodiment illustrative of the molecules disclosed herein isREGN727, also known in the art as Alirocumab selected from this list forpreparation and testing of cholestosome encapsulation according to theprinciples enumerated in Example 1. The particular preparation wasdesigned for oral use with exposure approximately 100 mg per month oftreatment.By way of specific example, REGN727 loaded cholestosomes with meandiameter of 250-450 nm can be prepared in the manner of the presentinvention, as described in example 1. Cholestosomes containing REGN727will be prepared using a novel blend of two cholesteryl esters. Thisconstruct will be used lower LDL cholesterol. The construct will begiven in combination with statin drugs and optionally in combinationwith ileal brake hormone releasing substances.

Statin Component of Combination Product

In order to raise HDL cholesterol and lower total cholesterol, the oralREGN727 will be co-formulated with an immediate release statin drug. Alisting of statins suitable for combination with oral PCSK-9 treatmentincludes the following: lovastatin, atorvastatin, rosuvastatin,simvastatin, fluvastatin, pitavastatin, pravastatin. By way of example a10 mg dose of atorvastatin is preferred but the invention of thecombination is not limited only to atorvastatin as most of the availablestatin molecules will be suitable, as all are immediate releaserequiring only film-coating.

Brake Component of PCSK-9 Combination Product

In order to lower triglycerides, the formulation of REGN727 and statinwill optionally be combined with approximately 10 grams of an ilealbrake hormone releasing substance as disclosed in US2011/0268795, thecomplete contents of and complete formulation of which are herebyincorporated by reference. This formulation releases the contents of theactive ileal brake hormone releasing substance at the ileum of thesubject, and completely controls elevated triglyceride concentrations.The results of studies performed by the inventors show that chronicdaily stimulation of the ileal hormones with Aphoeline Brake™, delivereddirectly into the ileum, tends to stabilize and maintain the bodyhomeostasis, as well as decrease in the fasting state the abnormallevels of insulin, glucose, triglycerides and all of the measured liverenzymes. Also the significant decrease in alpha-fetoprotein seems toindicate a decrease in inflammation of the liver. Combining the decreasein insulin resistance, triglyceride and liver inflammation with decreasein liver enzymes indicates a significant improvement in liver health andsignals a role for these hormones to play in regeneration of hepatocytesand maintaining liver health. Combining these beneficial properties witha Statin and a PCSK9 monoclonal antibody offers patients a novel andcomprehensive approach to control of metabolic syndrome, which is aprimary underlying cause of hyperlipidemia and the resultingatherosclerotic vascular disease.

The combination product resulting from these elements would beadministered to patients with hyperlipidemia on a once daily basis, withthe end result being a complete control of hyperlipidemia with minimalside effects.

Example 8 Use of Cholestosomes and Chylomicrons for Delivery of siRNAGenetic Material

In classical genetics, in a sexually reproducing organism (typicallyeukarya) the gamete has half the number of chromosomes of the somaticcell and the genome is a full set of chromosomes. The halving of thegenetic material in gametes is accomplished by the segregation ofhomologous chromosomes during meiosis. Any material derived from eitherfull or haploid chromosomes is genetic material.

The term genome can be applied specifically to mean what is stored on acomplete set of nuclear DNA (i.e., the “nuclear genome”) but can also beapplied to what is stored within organelles that contain their own DNA,as with the “mitochondrial genome” or the “chloroplastgenome”.Additionally, the genome can comprise non-chromosomal genetic elementssuch as viruses, plasmids, and transposable elements.

RNA and short chain RNA interference or insertions meant to alterfunctions of RNA are also considered genetic material for purposes ofencapsulation into cholestosomes and for purposes of delivery of geneticmaterials to sites inside target cells.

By way of example we disclose a combination approach to the treatment ofHepatitis C, an RNA virus of genus Flaviviridae. Members of this genushave monopartite, linear, single-stranded RNA genomes of positivepolarity, 9.6 to 12.3 kilobase in length. The 5′-termini of flavivirusescarry a methylated nucleotide cap, while other members of this familyare uncapped and encode an internal ribosome entry site. Virus particlesare enveloped and spherical, about 40-60 nm in diameter. Although over60 viruses in this genus are known to cause disease, we wish to focusattention on Genus Hepacivirus (type species Hepatitis C virus)

Hepatitis C is a particularly interesting target for cholestosometherapy because this virus hides in the normally observed lipidparticles and it appears necessary to follow the virus into these hidingsites if one wishes to interfere with its life cycle, invasiveness orpassage between individuals.

These latter goals will lead to our preparation of specific constructsuseful for the treatment of hepatitis C infections

A preferred embodiment illustrative of the molecules disclosed herein ismiR-122, known in the art as Miravirsen. By way of non-limiting example,alternative genetic constructs against Hepatitis C and other viruses maybe used as alternative treatments against the respective viruses, aslong as there is a need for a novel means of gaining access tointracellular sites and additionally to other circulating lipidparticles such as chylomicron remnants which are also known to shelterthe Hepatitis C virus.

miR-122 was selected for preparation and testing of cholestosomeencapsulated genetic materials targeting Hepatitis C, according to theprinciples enumerated in Example 1. The particular preparation wasdesigned for oral use and intracellular delivery, upon knowledge andbelief that Hepatitis C infected cells are a necessary intracellulartarget for a genetic modifying strategy against this virus. Even withless than optimal delivery, there is clinical evidence of effectiveresponse of Hepatitis C viral infections to treatment with miR-122constructs given parenterally to patients. These results are presentedbelow.

By way of specific example, miR-122 cholestosomes with mean diameter of250-10,000 nm can be prepared in the manner of the present invention, asdescribed in Example 1, with cholesteryl ester selection from the estersdisclosed as preferred in Example 2. Cholestosomes containing miR-122are prepared using a novel blend of two cholesteryl esters.

miR-122 for HepC in Cholestosomes

The stability and propagation of hepatitis C virus (HCV) is dependent ona functional-interaction between the HCV genome and liver-expressedmicroRNA-122 (miR-122). MicroRNAs are small non-coding RNAs encoded bythe human genome that transcriptionally and post-transcriptionallymodify gene expression. The microRNA-122 (miR-122) forms the dominantmicroRNA in the liver and is exclusively expressed in hepatocytes. Ithas been implicated in multiple different processes, including lipidmetabolism, cell differentiation, iron metabolism and hepatic circadianregulation. The 50 untranslated region (UTR) of HCV is highly conservedacross genotypes and contains two miR-122 binding sites, disruption ofwhich blocks HCV replication.

Miravirsen is a locked nucleic acid-modified DNA phosphorothioateanti-sense oligonucleotide that sequesters mature miR-122 in a highlystable heteroduplex, thereby inhibiting its function. As a 15 nucleotidelong oligonucleotide complementary to miR-122, miravirsen can formstable heteroduplexes with miR-122. Whether miravirsen exerts itsantiviral effects predominantly through sequestration of availablemiR-122, indirectly through disrupting lipid pathways essential to theviral lifecycle, or through other mechanisms remains under activeinvestigation.

Its efficacy against chronic HCV infection was first shown in studies inchimpanzees, the only natural HCV animal model. Chimpanzees thatreceived the highest, 5 mg/kg, dose through a weekly infusion had amarked decrease in plasma and liver HCV RNA, which led to clinicaltesting of miravirsen. Janssen and colleagues reported their findingsfrom a phase 2a study in treatment naive non-cirrhotic patientschronically infected with HCV genotype 1. They enrolled 36 patients whowere randomized to 5 weekly subcutaneous injections with three differentdoses of miravirsen (3, 5 or 7 mg/kg) or placebo. They found that HCVRNA showed a dose-dependent decline, with 1 (11%) patient in the 5 mg/kgand 4 (44%) patients in the 7 mg/kg groups reaching undetectable HCV RNAlevels, all after the fifth dose of miravirsen. Notably, the individualresponse curves shown by the authors were quite variable, even with thehighest dose. Three of the patients whose HCV RNA became undetectablerelapsed 4-5 weeks later and one patient went on to be treated withPegIFN/RBV. The long term outcome in the remaining patient who achievedan undetectable HCV RNA at study week 14 and remained undetectablethrough week 18 was not reported. Adverse events were generally mildwith only injection site reactions being likely related to miravirsenadministration.

The most likely explanation for the rather weak and variable response ofHCV patients to miravirsen is irregular cellular uptake of miravirsen.This is not surprising in that failure to reach the cellular targetcomplicates most attempts to commercialize antisense therapy. Poorintracellular penetration is the likely reason why the dose of themiR-122 formulation was 7 mg/kg. Effective intracellular delivery of theconstruct using cholestosomes could lower the effective dose to values10-100× lower. Additionally, there would be the advantage of oral use ina lower overall dosage than currently employed for use parenterally.

Cholestosome formulations will be made for the current miravirsenconstruct, and the likely result of successful formulation will be adramatically improved action on HCV viral load because of effectiveintracellular delivery. In addition, the cholestosome formulation willbe used orally, which is a great improvement over subcutaneousinjection. The unique feature of oral uptake of cholestosome-miR-122would be complemented by intracellular delivery of cholestosome-miR-122,which would make the product effective at a lower dose. Thesenanoparticles would enter cells via chylomicron loading, and once insidesilence the Hepatitis C virus.

There is much development work to be accomplished to successfullycommercialize miR-122 antisense technology. While the impact of oraltherapy with cholestosome encapsulated miR-122 will produce a muchimproved version of this construct, it is likely that the oralformulation will be co-administered with anti-HCV drugs such assofosbuvir (Sovaldi), and these combinations are claimed for use intreatment of HCV infections.

Furthermore, there is additional likelihood that anti-HCV drug therapywill be improved by concomitant use of Brake formulations to manageconcomitant metabolic syndrome manifestations, and in principle torepair and regenerate the liver of these patients, as was furtherdetailed in Example 7 with reference to hyperlipidemia.

With respect to the use of Brake in combination regimens for HepatitisC, the details of these formulations and strategy are found in WO2013/063527, published May 2, 2013, WO/2012-118712 A2, published Sep. 7,2012 and US2012 026561, the contents of which are herein incorporated byreference.

Accordingly, the ideal combination disclosed for management of HCV inall types of patients would be oral cholestosome-miR-122 combined withoral sofosbuvir, combined with oral Brake.

An example of an oral vaccine for HCV is provided in Example 9, and thisvaccine could be given to the same HCV patients as defined in thepresent example.

Gene Editing in the Treatment of HIV Viremia

The ability to make site-specific modifications to (or “edit”) the humangenome has been an objective in medicine since the recognition of thegene as the basic unit of heredity. The challenge of genome editing isthe ability to generate a single double-strand break at a specific pointin the DNA molecule. Numerous agents, including meganucleases,oligonucleotides that form DNA triplexes, and peptide nucleic acids,have been tested and shown to be limited by inefficiency. Another classof reagents, the zinc-finger nucleases (ZFNs), have proved versatile forgenome editing, and the use of ZFNs is now well established in a numberof model organisms and in human cells.

ZFNs are well suited for genome engineering because they combine the DNArecognition specificity of zinc-finger proteins (ZFPs) with the robustbut restrained enzymatic activity of the cleavage domain of therestriction enzyme FokI (a nuclease). ZFPs, which provide DNA-bindingspecificity, contain a tandem array of Cys₂His₂ zinc fingers, eachrecognizing approximately 3 base pairs of DNA. By comparison, thebacterial type IIS restriction endonuclease, FokI, has no sequencespecificity and must dimerize to cut the DNA. After the ZFN-mediateddouble-strand cut, the DNA can be repaired by either homologousrecombination or nonhomologous end joining. Homologous recombinationrepairs the break while preserving the original DNA sequence. However,these cells are susceptible to recutting by ZFNs. In contrast,nonhomologous end joining can result in random insertion or deletion ofnucleotides at the break site, resulting in permanent disruption of theprimary DNA sequence. Therefore, nonhomologous end joining can beexploited to mutate a specific gene, leading to its functional knockout.

The design of a ZFN pair consisting of two 4-finger proteins that bindto a target site within the human chemokine (C-C motif) receptor 5 gene(CCR5) was reported previously. In preclinical tests, CCR5-modified CD4T cells expanded and functioned normally in response to mitogens, wereprotected from human immunodeficiency virus (HIV) infection, and reducedHIV RNA levels in a humanized mouse model (involvingxenotransplantation) of HIV infection.

Tebas and colleagues selected CCR5, which encodes a coreceptor for HIVentry, for several reasons. First, its disruption seemed likely toincrease the survival of CD4 T cells; persons homozygous for a 32-bpdeletion (delta32/delta32) in CCR5 are resistant to HIV infection. Invitro, CD4 T cells from such persons are highly resistant to infectionwith CCR5-using strains of HIV, which are the dominant strains in vivo.Moreover, persons who are heterozygous for CCR5 delta32 and who have HIVinfection have a slower progression to the acquired immunodeficiencysyndrome. Furthermore, the effectiveness of blocking or inhibiting CCR5with the use of small-molecule inhibitors has been shown in humans.Finally, one person who underwent allogeneic transplantation withprogenitor cells homozygous for the CCR5-delta32 deletion has remainedoff antiviral therapy for more than 4 years, with undetectable HIV RNAand proviral DNA in the blood, bone marrow, and rectal mucosa. Althoughthe mechanism responsible for the apparent cure associated with thisprocedure remains to be established, acquired CCR5 deficiency is onepossibility. Tebas now reports the partial induction of acquired geneticresistance to HIV infection after targeted gene disruption (i.e., theinfusion of autologous CD4 T cells modified at CCR5 by a ZFN).

The ZFN in this case was given in association with an adenoviral vector,and cells were removed from the body prior to transfection. In the workof the inventors, overcoming these deficiencies with a functionalconcentration of the ZFN inside cells is feasible with a cholestosomeformulation.

By way of specific example, ZFN constructs active against CCR5 incholestosomes with mean diameter of 250-10,000 nm can be prepared in themanner of the present invention, as described in Example 1, withcholesteryl ester selection from the esters disclosed as preferred inExample 2. Cholestosomes containing ZFN are prepared using a novel blendof two cholesteryl esters selected to achieve a neutral or slightlynegatively charged particle that will be taken up by enterocytes anddeposited into chylomicrons.

This listing is provided by way of example of genetic materialpolynucleotides in cholestosome formulations across the range ofmolecule size in use for disease modification and treatment, and is inno way limiting on the application of cholestosomes for encapsulation ofmolecules of all sizes and when used for therapy of known or newdiseases.

Example 9 Use of Cholestosomes and Chylomicrons Therefrom for OralVaccination of Virus Infections that Hide Inside Cells Transmission LifeCycle of Hepatitis C Virus

Hepatitis C virus (HCV) interacts with apolipoproteins B (apoB) and E(apoE) to form infectious lipoviral particles. Response topeg-interferon is influenced by interferon-stimulated genes (ISGs) andIL28B genotype. LDL cholesterol (LDL-C) also predicts interferonresponse.

Hepatitis vaccines may be including adjuvants and miRNA or siRNA in thecase of Hepatitis C, a virus that hides in Lipid particles. A suitableantisense therapy example from Example 8 is miR-122. It is notable thatmiR-122 does not elucidate a response from the immune system, and infact it is notable that Hepatitis C does not elucidate a response fromthe immune system either. This is why it is so difficult to remove.

An Effective Hepatitis C Vaccine

An effective Hepatitis C vaccine will need to follow the virus thru thelipid pathway and create an immunological recognition of its presence inthe cells and perhaps in the lipid particles themselves. Accordingly theuse of an orally absorbed cholestosome formulation that places a vaccineconstruct into chylomicrons for delivery is a novel approach tovaccination.

There is no vaccine that follows the virus into all body cells, so theadaptation of the Hepatitis C viral construct intocholestosome-chylomicron delivery will be the first to use the lifecycleof a chronic infection virus against the organism directly. Use of aconcomitant adjuvant will also be an optional but necessary component ofthe oral cholestosome Hepatitis C vaccine construct. This vaccine willbe orally absorbed at the duodenum.

Oral Vaccines in Cholestosomes with or without Adjuvants

This same approach of an oral cholestosome encapsulated vaccine withadjuvant can be used for other chronic viral infections where the virushides inside body cells, to include HIV which hides in T-lymphocytes,Herpes zoster which hides in neural tissue, and other flavivirusconstructs with similar properties to hepatitis viruses.

It will also be a second preferred embodiment to deliver Hepatitis Cvaccine with adjuvants orally to the Peyer's patches dendritic cells ofthe ileum, and for this our ileal vaccine releasing technology will beemployed, as disclosed fully in PCT/US2013/031483, published asWO2013/148258 Mar. 10, 2013. Note that the disclosed vaccine there isnot a cholestosome formulation, and in fact we are not anticipating thatthe Hepatitis C vaccine construct delivered to the ileum would be orallyabsorbed, nor is this perceived as a requirement for efficacy.

Thus there is potential for a novel combination product in thisdisclosed example, one vaccine component which is cholestosome based topenetrate into the lipid pathways of the body and which modifies thevirus replication steps (and when combined with a drug, kills HepatitisC virus directly), and a second ileal targeted therapeutic vaccine whichtriggers a response in dendritic T-lymphocytes in Peyer's patches whereT lymphocytes are functioning as dendritic cells.

Use of these Hepatitis C vaccines in conjunction with Brake is optionedwhen the patient is in need of repair of fatty liver disease and earlycirrhosis, which offers maximum benefit to the patient with Hepatitis Cinfection. Brake therapy has been disclosed in Example 7 and isincorporated herein in combination with Hepatitis C vaccines deliveredby cholestosomes and delivered to the ileum for action on dendriticcells. These products may also be used in conjunction with anti-viralcompounds such as sofosbuvir to reduce viral load

Example 10 Use of Cholestosomes for Topical Delivery of Proteins forWound Healing, Infections and Inflammation and for Topical Delivery ofCosmetics

In the present invention, molecules used by IV injection for thetreatment of infectious diseases would be generally suitable forencapsulation into cholestosomes and used topically as an ointment orcream.

Most antibiotics disclosed in example 3 need to be injectedintravenously (IV), as the molecules are typically hydrophilic and nototherwise orally absorbed. Thus use in cholestosomes would enable theirabsorption into outer epidermidis. Numerous other small and largermolecules may be used in cholestosomes and administered topicallyaccording to the present invention including anti-fungals, anti-virals,anti-cancer and protein and peptide molecules used as growth factors.

There are many topical uses for treatments of disease that are enabledby cholestosome encapsulation of molecules. Some non-limiting examplesinclude wound healing with topical platelet derived growth factors toinclude combination with other growth factors known to be beneficial towound healing in the art.

An additional example would be the topical use of anti-TNF antibodiessuch as adalimumab (Humira) or Infliximab (Remicade) or many othersimilar molecules used topically for psoriasis and other dermalinflammatory diseases where these products are given currently bysubcutaneous injection. Nearly 4.1 million people were diagnosed withsome form of moderate-to-severe psoriasis in 2013. This number isexpected to climb slightly to 4.4 million by 2020, with 1.5 million ofthe population being treated with systemic agents. A rise in the globalprevalence of psoriasis, as well as an increase in the diagnosis rateresulting from improved diagnostic methods, will increase the demand forinjectable monoclonal antibodies but also justify more of these productsin topical cholestosome applications. As psoriasis is increasingly beingrecognized as a serious systemic disease with associated quality of lifeimpairment and disability, rather than as a simply cutaneous disease,healthcare professionals will consider cholestosome encapsulatedproteins and peptides as preferred over the older sub-optimaltreatments. Topical administration of currently injected vaccines wouldalso be facilitated by cholestosome formulations and the examplesprovided in Example 9 and previous prior art of the inventors areincluded here as non-limiting examples

None of these molecules are orally absorbed in the native state, and ineach case oral absorption would constitute a major advantage over thecurrent need to inject them parenterally. They could also be used in thetreatment of localized areas of disease thereby avoiding completely theside effects of drugs given systemically by injection.

Tobramycin for Treatment of Dermal Infections

A preferred embodiment illustrative of the molecules disclosed herein istobramycin, selected from this list for preparation and testing ofcholestosome encapsulated tobramycin according to the principlesenumerated in Example 1. The particular preparation was designed fororal use, and for increasing the overall action of the antibiotictobramycin against target gram negative bacteria such as Pseudomonasaeruginosa. A preparation of topical tobramycin might effectivelycontrol the pseudomonas diseases malignant otitis externa or be inhaledto effectively control pseudomonas in patients with cystic fibrosis.

By way of specific example, tobramycin cholestosomes with mean diameterof 250-1,000 nm were prepared in the manner of the present invention, asdescribed in Example 1, with cholesteryl ester selection from the estersdisclosed as preferred in Example 2. Cholestosomes containing tobramycinwere prepared using a novel blend of two cholesteryl esters, cholesterylmyristate and cholesteryl laurate.

Cholesteryl Esters Facilitate Skin Delivery

The ability of cholestosome encapsulated molecules to function incosmetic applications is an expected discovery within the art.

Major lipids are ceramides, cholesterol and free fatty acids. Thesecomponents of the stratum corneum lipid matrix play a key role inmammalian skin barrier function.

The effect of the cholesterol esters on the penetration of the stratumcorneum in vivo and in vitro were studied in by Kravchenko andcolleagues in rats and mice, and the effect of cholesterol esters on thefluidity of the liposome's lecithin were studied by the fluorometricmethod.

This study shows that inclusion of cholesterol esters to thistransdermal delivery system (TDS) increased the permeability of thestratum corneum for phenazepam. They observed the maximal fluidizationof the lipid environment in the presence of cholesteryl laurate,cholesteryl pelargonate, cholesteryl undecylate and cholesterylcapronate. Thus, cholesterol esters were found to be effective enhancersfor transdermal delivery, and lead to the current uses as disclosedherein.

Topical Use of Curcumin for Melanoma

The cholestosome formulation of curcumin difluoride (CDF) as disclosedin example 5 may also be useful topically for treatment of dermalcancers.

Previous work with liposomes and curcumin by Chen 2012 investigated thein vitro skin permeation and in vivo antineoplastic effect of curcuminby using liposomes as the transdermal drug-delivery system. Soybeanphospholipids (SPC), egg yolk phospholipids (EPC), and hydrogenatedsoybean phospholipids (HSPC) were selected for the preparation ofdifferent kinds of phospholipids composed of curcumin-loaded liposomes:C-SPC-L (curcumin-loaded SPC liposomes), C-EPC-L (curcumin-loaded EPCliposomes), and C-HSPC-L (curcumin-loaded HSPC liposomes). The physicalproperties of different liposomes were investigated as follows: photoncorrelation spectroscopy revealed that the average particle sizes of thethree types of curcumin-loaded liposomes were 82.37±2.19 nm (C-SPC-L),83.13±4.89 nm (C-EPC-L), and 92.42±4.56 nm (C-HSPC-L), respectively. Theencapsulation efficiency values were found to be 82.32±3.91%,81.59±2.38%, and 80.77±4.12%, respectively. An in vitro skin penetrationstudy indicated that C-SPC-L most significantly promoted drug permeationand deposition followed by C-EPC-L, C-HSPC-L, and curcumin solution.Moreover, C-SPC-L displayed the greatest ability of all loaded liposomesto inhibit the growth of B16BL6 melanoma cells. Therefore, the C-SPC-Lwere chosen for further pharmacodynamic evaluation. A significant effecton anti-melanoma activity was observed with C-SPC-L, as compared totreatment with curcumin solution in vivo. These results suggest thatC-SPC-L would be a promising transdermal carrier for curcumin in cancertreatment.

This example of topical treatment of cancer using a cholestosomepreparation of Curcumin difluoride CDF should not be consideredlimiting, and any of the anti-cancer compounds disclosed in example 5should be suitably enabled for topical use by encapsulation intocholestosomes.

Example 11 Use of Cholestosomes for Topical Delivery into the Lung

In the present invention, molecules used by IV injection for thetreatment of infectious diseases would be generally suitable forencapsulation into cholestosomes and used for inhalation, where thedelivery by cholestosomes would be expected to enhance penetration ofthe encapsulated compound into the cells lining the alveoli and bronchi.This is novel over prior art use of liposomes, which would not penetratecells, rather serving only to hold the compound in liposomes at the sitefor a longer period of time without enhancing cellular penetration.

Thus this pathway of delivery by aerosolization of cholestosomeencapsulated nanoparticles is rational and may greatly enhance efficacyin the treatment of pulmonary diseases such as asthma, COPD, lungcarcinoma, cystic fibrosis, and even rare conditions such as Alpha-oneAnti-trypsin deficiency

Most antibiotics disclosed in example 3 need to be injectedintravenously (IV), as the molecules are typically hydrophilic and nototherwise orally absorbed. Thus use in cholestosomes by inhalation wouldenable their absorption into lung directly via their enhanced cellularpenetration mechanisms disclosed herein. Numerous other small and largermolecules may be used in cholestosomes and administered by inhalationaccording to the present invention including anti-fungals, anti-virals,anti-cancer and protein and peptide molecules used as growth factors.

There are many Pulmonary disease applications to disease treatmentenabled by cholestosome encapsulation of molecules. Some non-limitingexamples include repair of viral or chemical burn damage to lung alveoliwith platelet derived growth factors to include combination with othergrowth factors known to be beneficial to wound healing in the art.

It is noted that very small nanoparticles will be needed for inclusionof cholestosome encapsulated molecules in inhalers, probably smallerthan 100 nm for this application. Some non-limiting examples ofcompounds used in liposomes are offered as a proof of concept and aroadmap for improved intracellular delivery in the lung via cholestosomeencapsulation:

Iloprost Example:

Kleemann et al Pharm Res 2007: Pulmonary arterial hypertension (PAH) isa severe and progressive disease. The prostacyclin analogue iloprost iseffective against PAH, but requires six to nine inhalations per day. Thefeasibility of liposomes to provide a sustained release formulation toreduce inhalation frequency was evaluated from a technological point ofview.

Liposomal formulations consisting of di-palmitoyl-phosphatidyl-choline(DPPC), cholesterol (CH) andpolyethyleneglycol-di-palmitoyl-phosphatidyl-ethanolamine (DPPE-PEG)were prepared. Their physico-chemical properties were investigated usingdynamic light scattering, atomic force microscopy and differentialscanning calorimetry. Stability of liposomes during aerosolization usingthree different nebulizers (air-jet, ultrasonic and vibrating mesh) wasinvestigated with respect to drug loading and liposome size, pre- andpost-nebulization.

The phospholipid composition affected the diameters of liposomes onlyslightly in the range of 200-400 nm. The highest iloprost loading (12mcg/ml) and sufficient liposome stability (70% drug encapsulationpost-nebulization) was observed for the DPPC/CH (70:30 molar ratio)liposomes. The formulation's stability was confirmed by the relativelyhigh phase transition temperature (53 degrees C.) and unchanged particlesizes. The incorporation of DPPE-PEG in the liposomes (DPPC/CH/DPPE-PEG,50:45:5 molar ratio) resulted in decreased stability (20-50% drugencapsulation post-nebulization) and a phase transition temperature of35 degrees C. The vibrating mesh nebulizer offered a number ofsignificant advantages over the other nebulizers, including theproduction of small aerosol droplets, high output, and the lowestdeleterious physical influence upon all investigated liposomes.

Iloprost-loaded liposomes containing DPPC and CH components yieldformulations which are well suited to aerosolization by the vibratingmesh nebulizer.

The use of 200-400 nm size liposomes is probably too large forsuccessful commercial development

Salbutamol

Elhissi A M et al. J Pharm Pharmacol. 2006; 58:887-94. Multilamellar andoligolamellar liposomes were produced from ethanol-based soyaphosphatidyl-choline proliposome formulations by addition of isotonicsodium chloride or sucrose solutions. The resultant liposomes entrappedup to 62% of available salbutamol sulfate compared with only 1.23%entrapped by conventionally prepared liposomes. Formulations wereaerosolized using an air-jet nebulizer (Pari LC Plus) or avibrating-mesh nebulizer (Aeroneb Pro small mesh, Aeroneb Pro largemesh, or Omron NE U22). All vibrating-mesh nebulizers produced aerosoldroplets having larger volume median diameter (VMD) and narrower sizedistribution than the air-jet nebulizer. The choice of liposomedispersion medium had little effect on the performance of the Parinebulizer. However, for the Aeroneb Pro small mesh and Omron NE U22, theuse of sucrose solution tended to increase droplet VMD, and reduceaerosol mass and phospholipid outputs from the nebulizers. For theAeroneb Pro large mesh, sucrose solution increased the VMD of nebulizeddroplets, increased phospholipid output and produced no effect onaerosol mass output. The Omron NE U22 nebulizer produced the highestmass output (approx. 100%) regardless of formulation, and the deliveryrates were much higher for the NaCl-dispersed liposomes compared withsucrose-dispersed formulation. Nebulization produced considerable lossof entrapped drug from liposomes and this was accompanied by vesiclesize reduction. Drug loss tended to be less for the vibrating-meshnebulizers than the jet nebulizer. The large aperture size mesh (8 mum)Aeroneb Pro nebulizer increased the proportion of entrapped drugdelivered to the lower stage of a twin impinger. This study hasdemonstrated that liposomes generated from proliposome formulations canbe aerosolized in small droplets using air-jet or vibrating-meshnebulizers. In contrast to the jet nebulizer, the performance of thevibrating-mesh nebulizers was greatly dependent on formulation. The highphospholipid output produced by the nebulizers employed suggests thatboth air-jet and vibrating-mesh nebulization may provide the potentialof delivering liposome-entrapped or solubilized hydrophobic drugs to theairways.

Cholestosome Formulations for Inhalation

Target compounds for encapsulation in 100 nm or smaller cholestosomesand used by aerosol delivery include tobramycin for cystic fibrosisinfections, curcumin difluoride for lung carcinoma, siRNA for lungcarcinoma, vancomycin for pneumonia caused by MRSA, Ceftaroline forpneumonia caused by MRSA, fosfomycin for gram negative pneumonia.

Mepolizumab for Eosinophilic Asthma

A recently developed monoclonal antibody under clinical development is afurther example of an inhaled cholestosome formulation of a monoclonalantibody. Mepolizumab, an investigational, fully humanized IgG1IL-5-specific monoclonal antibody, met its primary endpoint in two PhaseIII studies of patients with severe eosinophilic asthma who did not seea reduction in exacerbations with high-dose inhaled corticosteroids andan additional controller drug. In the double-blind, parallel-group,multicenter, placebo-controlled, randomized MEA115588 study, 576patients ages 12 and older were given either 75 mg of intravenousmepolizumab or 100 mg of subcutaneous (SC) mepolizumab every four weeksover a total period of 32 weeks. Some 47 percent of patients in the75-mg IV treatment arm, and 53 percent of patients in the 100-mg SCtreatment arm met the study's primary endpoint of reductions inexacerbations. In the second double-blind, parallel-group, multicenter,placebo-controlled, randomized study, known as MEA115575, 135 patientsages 12 and older were given 100 mg of SC mepolizumab every four weeksover a total period of 24 weeks. This study met its primary endpoint ofreducing oral corticosteroid use while maintaining asthma control duringweeks 20-24. The company plans to file for regulatory approval formepolizumab, which would also continue the investigational developmentof mepolizumab in COPD and eosinophilic granulomatosis withpolyangiitis.

Clearly, an inhaled cholestosome formulation of mepolizumab would be aviable alternative to subcutaneous injection with this product, and theintracellular penetration may allow the dosage requirements to bedecreased by 10-100 fold over the current requirements of 100 mg once amonth. Accordingly, it is one preferred embodiment to develop acholestosome formulation of approximately 5 mg of this monoclonalantibody for inhalation use. In addition to lower dosage requirements,the topical use of this product by inhalation would produce an immediateresponse in patients in need thereof, and would thereafter beneficiallylower systemic exposure to a potent suppressive agent against theeosinophilic immune response, protective against a host of parasiticinvaders.

This listing is provided by way of example of inhaled cholestosomeformulations of known molecules across the range of molecule size incommon use for disease treatment, and is in no way limiting on theapplication of cholestosomes for encapsulation of molecules of all sizesand when used for inhalation therapy of pulmonary diseases.

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1-84. (canceled)
 85. A pharmaceutical composition comprising at leastone pharmaceutically active agent which is encapsulated in the core of ananoparticle vesicle, wherein said vesicle outer surface is comprised ofone or more cholesteryl esters and said nanoparticle outer surfacecoating of cholesteryl esters is not broken by cholesterol transporterson the surface of duodenal enterocytes during absorption, such that saidnanoparticle enters said duodenal enterocyte as an intact vesicle andsaid duodenal enterocyte adds said intact vesicle into its chylomicronsfor transportation of said active pharmaceutical composition to bodycells in need thereof.
 86. The composition of claim 85, where saidpharmaceutically active agent(s) is not detected by cells during passageof said vesicle through the cell membranes of said cells, saidcomposition and cellular intake process resulting in the production of atransformed chylomicron whereby said transformed chylomicron cantransport said active pharmaceutical composition into cells
 87. Thecomposition of claim 86 wherein said pharmaceutical composition in thecore of said nanoparticle vesicle is released by the action ofintracellular cholesterol hydrolase enzymes on said vesicle surfacecholesteryl esters and said molecule is released inside cells intact.88. The composition of claim 85 wherein said vesicle surface layer isformed from one or more non-ionic cholesteryl esters produced fromcholesterol and at least one C₄-C₃₆ fatty acid, wherein the mass ratioof the pharmaceutically-active agent to one or more cholesteryl estersis between about 4:96 to about 96:4, said nanoparticle being capable ofdelivering said pharmaceutically active agent within cells of a patientor subject to a concentration level at least 2 times the levelobtainable in the absence of said nanoparticle vesicle.
 89. Thecomposition according to claim 85 in oral dosage form, wherein afteroral administration to a patient or subject the cholesteryl esters reactwith cholesterol transporters on the surface of duodenal enterocytes andload said vesicles into chylomicrons, resulting in oral absorption ofthe vesicles and the production of a transformed chylomicron which canbe transported in lymph channels and blood vessels to cells, whereuponsaid cells ingest said pharmaceutical composition and after cholesterolhydrolase action, said pharmaceutically active agent being released inthe cytoplasm of said cells
 90. The composition according to claim 85,wherein said composition is in a dosage form suitable for oraladministration.
 91. The pharmaceutical composition according to claim85, wherein the vesicle comprises as cargo a peptide which is selectedfrom the group consisting of a hydrophilic peptide, human growthhormone, prolactin, oxytocin, calcitonin, bovine growth hormone, porcinegrowth hormone, Ghrelin, GLP-1, PYY36, Oxyntomodulin, GLP-2, Glucagon,and any composition of insulin, including but not limited to regularinsulin, NPN insulin, Lente insulin, recombinant insulin, insulinglargine, insulin lispro, insulin degludec and wherein said vesicle isencapsulated by a surface layer consisting essentially of at least twocholesteryl esters.
 92. The pharmaceutical composition according toclaim 85 wherein the pharmaceutically active agent is a protein, apolypeptide, a polynucleotide, an antibiotic or an antiviral agent. 93.The pharmaceutical composition according to claim 85, wherein thesurface layer of the vesicle is enterically coated.
 94. Thepharmaceutical composition according to claim 85, wherein the surfacelayer of said vesicle remains intact at a pH range of between about 2 toabout
 14. 95. The pharmaceutical composition according to claim 85,wherein the vesicle has a diameter ranging from about 100 nm to 10,000nm.
 96. The pharmaceutical composition according to claim 85, whereinsaid vesicle has a neutral surface defined by a Zeta potential of about−40 to +20.
 97. The pharmaceutical composition according to claim 85,wherein the cholesteryl esters are selected using an interdigitatedalternating alkyl chain model which maximizes the mass ratio ofpharmaceutically-active agent to the one or more cholesteryl esters byselecting the one or more cholesteryl esters based onpharmaceutically-active agent-cholesteryl ester functional groupinteractions.
 98. The pharmaceutical composition according to claim 85,wherein the pharmaceutically active agent is selected from the groupconsisting of a composition of any hydrophilic peptide, any insulin,human growth hormone, prolactin, oxytocin, calcitonin, GLP-1, PYY36,Oxyntomodulin, GLP-2, Glucagon, interferon, curcumin di-fluoride,ceftaroline, KBP-1404, vancomycin, bevacizumab, trastuzumab, adalimumabor an anti-PCSK-9 monoclonal antibody.
 99. The composition according toclaim 96, wherein said GLP-1 comprises a composition modified to improvestability to DPP-IV enzymatic degradation or modified to prolong itscirculation time in the blood.
 100. The composition according to claim99 wherein said GLP-1 composition is selected from the group consistingof lixisenatide, exenatide, liraglutide, albiglutide, or derivativesthereof.
 101. The composition according to claim 85 wherein thepharmaceutically-active agent is an anti-hyperlipidemic monoclonalantibody and the pharmaceutical composition further comprises a statinand optionally an ileal brake hormone releasing substance.
 102. Thecomposition according to claim 85 wherein said vesicle is cargo-loadedwith an active agent which is unable to appreciably pass throughduodenal enterocyte cells in the absence of said cholesteryl esternanoparticle, said vesicle having a neutral surface and an ability topass into duodenal enterocytes of a patient or subject administered saidcargo-loaded nanoparticle in the manner of orally absorbed nutrientlipids and be incorporated into chylomicrons to produce a nanoparticleloaded chylomicron capable of carrying the pharmaceutically active agentinto cells.
 103. The composition according to claim 85 wherein saidactive agent is microRNA miR-122, any nucleotide sequence, DNA or RNA orfragments thereof.
 104. The composition according to claim 85 whereinsaid active agent is a vaccine and the vesicle optionally furtherincludes an adjuvant to enhance the immunogenic effect of the vaccine onthe immune system in a patient.
 105. A method of delivering apharmaceutically active agent to a target inside a cell of a patient orsubject, said method comprising administering to said patient or subjecta cargo-loaded vesicle according to claim 85, wherein the vesicle willbe taken up duodenal enterocytes within the patient or subject,incorporated into a chylomicron and the chylomicron will dock on a celland release its contents, including the active agent, within the cell,the concentration of active within the cell being substantially greaterthan when said agent is delivered to said cell using conventional means.106. The method according to claim 104 wherein said cholestosomedelivers active agent into a target cell in an amount at least 10 foldgreater than when the active agent is delivered to the cell in theabsence of said cholestosome.
 107. A pharmaceutical compositioncomprising a pharmaceutically active agent which is encapsulated in ananoparticle vesicle having a neutral surface comprising a corecontaining said pharmaceutically active agent and a surface layersurrounding said core and said agent, said surface layer consistingessentially of one or more non-ionic cholesteryl esters produced fromcholesterol and at least one C₄-C₃₆ fatty acid, wherein the mass ratioof the pharmaceutically-active agent to one or more cholesteryl estersis between about 4:96 to about 96:4, said nanoparticle being capable ofdelivering said active agent within cells of a patient or subject to aconcentration level at least 2 times the level obtainable in the absenceof said cholestosomes.
 108. The composition according to claim 107,wherein said composition is in a dosage form suitable for oral, topical,inhalation or vaginal administration.